Chemiluminescence enhancement by trypanocidal drugs and by inhibitors of antioxidant enzymes in Trypanosoma cruzi

Molecular and Biochemical Parasitology, 30 (1988) 243-252 Elsevier

243

MBP 01015

Chemiluminescence enhancement by trypanocidal drugs and by inhibitors of antioxidant enzymes in Trypanosoma cruzi C e c i l i a G i u l i v i , J u l i o F. T u r r e n s a n d A l b e r t o B o v e r i s Instituto de Quimica y Fi~'icoquimica Biol6gicas, Facultad de karmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina (Received 7 January 1988: accepted 27 April 1988)

The spontaneous emission of chemiluminescence by Trypanosoma cruzi epimastigotes was 133 ~ 5 counts s -I (mg protein) -I. The measured intracellular steady state concentration of hydrogen peroxide in the same cells was 1.5 ± 0.5 p,M. These two values are about 12- and 15-times higher than the corresponding ones for isolated rat hepatocytes. The intracellular steady state concentrations of superoxide radical and hydrogen peroxide were apparently increased by inhibiting superoxidc dismutase (with diethyldithiocarbamate or KCN addition) and by the addition of two different trypanocidal agents ([3-1apachone and nifurtimox) capable of intracellular redox cycling and in each case an increased chemiluminescence was observed. Depletion of intracellular reduced non-protein SH groups by 80% increased 3-fold the chemiluminescence of T. cruzi cells. It is apparent that both an increase in the intracellular steady state concentration of superoxide anion or hydrogen peroxide and a decrease in the level of reduced SH groups lead to an increase in the level of peroxy radicals which are the precursor species for light emission. Key words: Chemiluminescence; Oxygen radical; Antioxidant enzyme; Trypanosoma cruzi

Introduction

The epimastigotes of Trypanosorna cruzi, as well as most Trypanosomatids, have poor enzymatic defenses against intracellular hydroperoxides [1-3]. Catalase and Se-containing glutathione peroxidase are absent [3] and superoxide dismutase is present in relatively low proportion as compared to mammalian cells [3,4]. The hydroperoxide-catabolizing activities identified in T. cruzi are afforded by (a) an ascorbate peroxidase activity that uses hydrogen peroxide and (b) a nonspecific glutathione peroxidase activity able to utilize organic hydroperoxides [3]. A novel SHcontaining compound, trypanothione [5,6], may

Correspondence address: C. Giulivi, Instituto de Quimica y Fisicoquimica Biol6gicas. Facultad dc Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina. Abbreviations: D'FPAC, diethylenctriaminepentaacctic acid; GSH, glutathione.

eventually play a role in the peroxide metabolizing system of T. cruzi cells [7,8]. Cellular oxidative stress occurs if the intracellular steady state concentrations of the products of the partial reduction of oxygen, i.e. superoxide anion and hydrogen peroxide, are increased. Cellular damage associated with oxidative stress can be determined by several biochemical and biophysical methods. Based on its non-destructive character, low level chemiluminescence was used to detect the occurrence of electronically excited states derived from chain reactions initiated by the intermediates of oxygen reduction. Production of superoxide and hydrogen peroxide can lead by a chelated-iron catalyzed reaction to generation of hydroxyl radicals leading to a free radical chain reaction which in turn may generate excited species such as singlet oxygen and carbonyl compounds [9]. In this paper we report the effects of inhibitors of the antioxidant enzymes on the chemiluminescence of T. cruzi cells and the effect of two trypanocidal drugs (nifurtimox and 13-1apachone)

0166-6851/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

244

on superoxide production, intracellular hydrogen peroxide concentrations and chemiluminescence of T. cruzi epimastigotes. Materials and Methods

Biological materials. T. cruzi epimastigotes (Tulahuen 0 strain) were cultured in a liquid medium at 28°C, as previously described [10]. Cells were collected by centrifugation and resuspended in 145 mM NaCI, 6 mM glucose and 10 mM Tris-HCl, pH 7.4 (NaCl-glucose-Tris buffer) to a final concentration of 8-10 mg protein ml -~ Homogenates were prepared by freezing and thawing the cell suspensions three times, homogenizing the suspensions every time by passages through a syringe with a 25 gauge needle [4]. Chemiluminescence assay. Low level chemiluminescence was determined using a Packard scintillation counter model 332(I. The measurements were carried out using one of the photomultipliers by setting the scintillation counter in the •out-of-coincidence" modc and selecting a low thrcshold[ 11]. Trypanosomcs were suspended in 145 mM NaCI. (I.l mM diethylcnctriamincpentaacetic acid (DTPAC) and 20 mM Tris-HCl, pH 7.4 (NaCI-DTPAC-Tris buffer) and placed inside 10-ram-diameter and 35-mm-height glass tubes that were placed in scintillation vials made of lowpotassium glass [ 11 [. Biochemical analysis. The intracellular concentration of hydrogen peroxide in T. cruzi epimastigotes was determined by adapting an assay developed for soybean embryonic axes [12]. The cells were left during 1 h (or 15 min when trypanocidal drugs were used) in NaCI-DTPAC-TrisHCI buffer to equilibrate intracellular and extracellular concentrations of hydrogen peroxide. The incubation was stopped by addition of 1/10 of the volume of 100% (w/v) trichloroacetic acid, and neutralized to pH 7.4 with K O H 5 M. Hydrogen peroxide concentration in the supernatant was measured in 0.1-4).3 ml aliquots by formation of compound II using horseradish peroxidase at 417-402 nm in a dual wavelength spectrophotometer [13]. The specificity of the assay was provided by incubations of parallel samples with 10

~M catalase. The determinations were repeated with different amounts of epimastigotes reaching the same final hydrogen peroxide concentration in the supernatant, a condition that was taken as indication that hydrogen peroxide had reached a diffusion equilibrium with the external medium. Superoxide dismutase activity was assayed spectrophotometrically at 480 nm in a single beam spectrophotometer, determining the inhibition of 1 mM epinephrine autooxidation as described by Misra and Fridovich [14], in a reaction mixture consisting of 50 mM glycine-NaOH buffer, pH 10.2. Superoxide anion production was determined spectrophotometrically from the superoxide dismutase-sensitive rate of adrenochrome production at 480-575 nm (E = 2.97 mM -1 cm -~ [15]) in a dual wavelength 356 Perkin Elmer spectrophotometer. Total non-protein thiols groups were assayed fluorometrically in a MPF-3 Perkin-Elmer fluorometer using o-phthalaldehyde by the assay of Hissin et al. [16]. The 5,5-dithiobis(2-nitrobenzoic acid)-GSSG reductase assay was used for determining total cell glutathione (GSH + 1/2 GSSG) [17]. Oxygen consumption was measured by a Clark electrode in T. cruzi epimastigotes suspended in NaCI-DTPAC-Tris buffer. Malondialdehyde was determined by the thiobarbituric assay [18]. Aliquots of 1.5 ml of epimastigote suspensions were taken at 120 min of incubation and precipitated with 10% cold trichloroacetic acid, and centrifuged at 3000 rpm, for 5 min. Supernatants (1 ml) were incubated with 1 ml of thiobarbituric acid 0.67% (w/v) for 10 min at 100°C. The values were expressed as nmol of malondialdehyde (mg p r o t e i n ) 1 using an E = 156 mM -l cm -l at 535 nm [18]. All the incubations and determinations with T. cruzi epimastigotes and the measurements of enzyme activities were carried out at 28°C. Protein concentration was determined by the Folin reaction with serum albumin as standard [19]. Horseradish peroxidase and epinephrine were obtained from Sigma (St. Louis, MO). The trypanocidal agents nifurtimox and benznidazol were obtained from Bayer A.G. and Hoffmann-La

245

Roche Lab., respectively. 13-Lapachone was provided by Prof. J.D. Coussio (Dept. of Pharmacology, School of Pharmacy and Biochemistry, University of Buenos Aires). Other reagents were of analytical grade. The values given in tables and figures indicate mean values +-- S.E.M. Each point represents the mean of 5 experiments. Results

The dependence of spontaneous chemiluminescence upon protein concentration in suspensions of T. cruzi epimastigotes is shown in Fig. 1. For comparative purposes similar data on the spontaneous chemiluminescence of rat hepatocytes are included. Chemiluminescence increased almost linearly with protein concentration up to a maximum where turbidity started quenching the light emission. The slopes of the linear part, expressed in counts s -~ (mg protein) -~, allow the comparison between the two cell types, disregarding the differences in shape, mobility and light scattering. Thus, spontaneous emission of low level chemiluminescence by T. cruzi epimastigores was 133 -+- 5 counts s -1 (mg protein) -l, a

100

value 12-fold higher than the emission of isolated hepatocytes under the same experimental conditions (11 +- 2 counts s -1 (mg protein)-1). Our experimental conditions were fixed at 0.25 mg protein m1-1, giving maximal emission and the best signal-to-noise ratio. The obtained values of spontaneous chemiluminescence were 70 counts s t (mg protein) -1 and 13 counts s -I (mg protein) ~ for T. cruzi epimastigotes and hepatocytes, respectively. The measured intracellular steady state concentration of hydrogen peroxide in T. cruzi cells was 1.5 - 0.5 I~M (mean +- SEM), about 15-times higher than the value measured for hepatocytes using the same technique (0.1 txM). Addition of cyanide stimulated spontaneous chemiluminescence from T. cruzi up to 7-fold when added at concentrations higher than 1 mM (Fig. 2). A possible target for KCN could be the cyanide-sensitive superoxide dismutase activity previously detected in T. cruzi [4]. The superoxide dismutase activity of epimastigote homogenate was inhibited in more than 80% of its original activity (3 U (mg protein) -1) by KCN (Fig. 2). The KCN concentration that increased chemiluminescence at 50% of the maximal stimulation

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246

(0.72 mM) was close to the value that completely inhibited superoxide dismutase activity (1 mM). Other inhibitors of the CuZn superoxide dismutase such as azide and hydrogen peroxide were also effective in inhibiting the superoxide dismutase activity from 7-. cruzi epimastigotes. After 30 min of incubation of T. cruzi homogenates with 5 mM azide or with 2.8 mM hydrogen peroxide, superoxide dismutase activity decreased by 70% and 100%, respectively. Incubation at 100°C for 10 min also inhibited this activity (Table I). The copper chelator diethyldithiocarbamate also inhibited superoxide dismutase activity in epimastigote homogenate and stimulated chemiluminescence in the whole cells at concentrations above 1 mM (Table I and Fig. 3). The concentration of inhibitor that enhanced the emission of chemiluminescence to a maximum is related again to that which inhibited SOD activity. When reduced sulphydryl groups were blocked by addition of iodoacetamide to T. cruzi epimastigotes, the spontaneous chemiluminescence increased up to three-fold for iodoacetamide concentration above 30 I~M (Fig. 4). The increased chemiluminescence was accompanied by a decrease in the intracellular content of non-proteinSH groups, as determined by the o-phthalaldehyde method [16], from 4.1 nmol (rag protein)-1 to 0.8 nmol (mg protein)1 for iodoacetamide concentrations above 30 txM (Fig. 4). Total glutathione content was determined by the specific enzymatic assay of Tietze et al. [17], since trypanothione disulphide and other intermediates are not substrates for glutathione reductase [6,20].

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TABLE I Effect of inhibitors on the superoxide dismutase activity of T. cruzi

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Treatment

Inhibition of S O D activity (%)

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247

Reduced glutathione was slightly affected, decreasing from 0.5 nmol (mg protein) -1 to 0.38 nmol (mg protein) -~ in the same range of iodoacetamide concentrations (Fig. 4). The trypanocidal drugs 13-1apachone and nifurtimox, which were reported to be able to stimulate superoxide radical production through intracellular redox cycling [21-23], were added to intact epimastigotes to determine effect on chemiluminescence. Another trypanocidal agent, benznidazol, which due to its low redox potential does not seem to generate significant amounts of oxygen radicals [24], was also tested as a control. Addition of either 13-1apachone or nifurtimox to T. cruzi epimastigotes stimulated chemiluminescence 4.5- and 4-fold, respectively, for concentrations of about 10 txM (Fig. 5). Benznidazol did not significantly stimulate chemiluminescence up to 50 p,M. The effects of these drugs on chemiluminescence seem to reflect their capability to generate superoxide anion and hydrogcn pcroxide as previously described [21-24]. Oxygen consumption by T. cruzi epimastigotes was 11 nmol min-l (mg protein)-l; addition of 1 mM KCN reduced this value to 0.85 nmol min-t

(mg protein) ~. Addition of 13-1apachone and nifurtimox produced stimulation of the cyanide-insensitive respiration by T. cruzi epimastigotes. No increase in the cyanide-insensitive oxygen consumption was observed upon addition of benznidazol (Fig. 6). T. cruzi homogenates generated superoxide anion in the presence of NADH or NADPH (Table 1I). Addition of NADH or NADPH was essential in order to have measurable rates of superoxide production; NADH being about 1.6 times more effcctive than NADPH. Addition of 13-1apachone or nifurtimox to T. cruzi homogenates, supplemented with NADH or NADPH, stimulated superoxide anion production. No effect was observed upon benznidazol addition. The values of intracellular hydrogen peroxide concentration measured in epimastigotcs suspensions supplemented with the trypanocidal drugs are given in Table III. 13-Lapachone and nifurtimox increased 3- and 2.7-times the steady state concentration of hydrogen peroxide, whereas benznidazol did not modify hydrogen peroxide level. Fig. 7 shows the effect of 13-1apachone, nifurC

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Fig. 6. Effect of trypanocidal drugs on KCN-insensitive respiration of T. cruzi epimastigotes. Intact T. cruzi cells were supplemented with 1 mM KCN and with the trypanocidal drugs lS-lapachone (e), nifurtimox (A) and benznidazol (¢).

248 TABLE II

TABLE III

Superoxide production in 7". cruzi homogenates supplemented with 0.3 mM NADH or NADPH in the presence of trypanocidal drugs

Inlracellular steady state concentration of hydrogen peroxide by T. cruzi cells in the presence of trypanocidal drugs

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Addition (~.M)

(nmol min -l (mg protein) 1) NADH

NADPH

None

0.82 - 0.09

0.5 -+ 0.2

I~-Lapachone (50) Nifurtimox (50) Benznidazol (50)

5.7 ± 0.1 3.2 ~- 0.9 0.8 ± 0.2

0.9 -+ 0.1 1.4 ± 0.7 0.52± 0.05

timox and benznidazol on lipid peroxidation in T. by determining the amount of thiobarbituric acid-reactive material formed upon incubation. Similar concentrations of benznidazol were ineffective. cruzi cells, as measured

Discussion

A previous report indicated that the steady state concentration of oxygen radicals determines the

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HzO 2 concentration

(~M)

(~M)

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spontaneous emission of low level chemiluminescence by isolated hepatocytes [25]. In this study the same technique has been applied to T. cruzi epimastigotes to indirectly evaluate the intracellular concentration of oxygen radicals. The results indicate that T. cruzi epimastigotes maintain a relatively higher steady state of peroxy radicals which are the precursors of the emitting species and that derive from an elevated intracellular concentration of hydrogen peroxide and superoxide anion. The measured intracellular concentration of hydrogen peroxide in T. cruzi was 1.5 p.M, which is lower than that reported for T. brucei [26], and approximately one order of magnitude higher than that found in liver cells. Meshnick et al. [26] reported an intracellular concentration of hydrogen peroxide in T. brucei as high as 70 ~M, suggesting that increasing even more that hydrogen peroxide concentration could be used as a goal for designing trypanocidal agents. This unusually high hydrogen peroxide concentration reported in T. brucei has recently been questioned by several authors [8] including Meshnick [26]. In T. brucei trypomastigotes one would indeed expect a low intracellular concentration of hydrogen peroxide considering the high sensitivity of the L-alphaglycerophosphate dehydrogenase to hydrogen peroxide and the essential role of this enzyme in energy metabolism [27,28]. The higher intracellular concentration of hydrogen peroxide in T. cruzi epimastigotes as compared to hepatocytes is reflected in an increased chemiluminescence, 12-fold higher than that of isolated hepatocytes. Cyanide has different targets and with opposite effects on electron flow and superoxide anion and hydrogen peroxide metabolism. Cyanide at ~.M

249

concentration inhibits several hemoproteins (catalase and cytochrome oxidase). The action on cytochrome oxidase will result in inhibition of the production of superoxide anion by the autoxidation of ubisemiquinol [28,29] and in stimulation of the superoxide generation by NADH-dehydrogenase [30]. At higher concentrations (mM), cyanidc inhibits the CuZn-form of superoxide dismutase [31]. In this case, the effect of KCN on free radical metabolism could not be the consequence of catalase inhibition since this enzyme is missing in T. cruzi [3], neither could it be duc to changes in mitochondrial activities since the inhibition of the respiratory' chain only requires 0,M concentrations of KCN [13], The inhibition of superoxide dismutase by cyanide or Cu-chelators confirmed a previous report which indicated the presence of a KCN-sensitive superoxide dismutase activity [4]. It has been reported, however, that T. brucei, other strains of T. cruzi, Leishmania and Crithidia contain a Fe superoxide dismutase [32] and the CuZn isoenzyme has been reported as not present in several members of the Kinetoplastida [32]. Our results seem to indicate that a CuZn superoxide dismutase is present in the epimastigote form of T. cruzi (Tulahuen 0 strain). Further purification of this enzyme will be required in order to unequivocally identify a CuZn superoxide dismutase. It is also possible than a polypeptide bound to a transition metal may be mimicking a CuZn-SOD activity. The effect of superoxide dismutase inhibition on chemiluminescence points to superoxide anion as an important intermediate in the cascade of free radical reactions, possibly through an Fecatalyzed Haber-Weiss reaction [33,34]. A similar effect of KCN inhibiting superoxide dismutase and increasing spontaneous chemiluminescence has been reported in the unicellular eukaryote Acanthamoeba castellanii [35]. It should be pointed out that in the situation of superoxide dismutase inhibition, the only parameter of oxygen radical metabolism that is affected is the steady state concentration of superoxide radical which will vary inversely with residual enzyme activity. Spontaneous dismutation and remaining superoxide dismutase activity will be able to provide both a normal rate of formation and a normal steady state concentration of hydrogen per-

oxide. The role of the intracellular reduced SH groups in hydroperoxide metabolism and free radical chain reaction is evidenced by the effect of the suiphydryl reagent iodoacetamide on the emission of chemiluminescence (Fig. 4). The comparable values of iodoacetamide concentration needed to decrease the non-protein SH groups and required to increase T. cruzi chemiluminescence seem to indicate the importance of trypanothione and other related compounds in the maintenance of antioxidant defenses in T. cruzi. The role of glutathione seems to be secondary since no changes in its concentration were observed simultaneous to important changes in chemiluminescence. It seems likely that reduced trypanothione may be acting as an effective breaker of the free radical chain reactions, although a role of reduced trypanothione as hydrogen donor for both trypanothione and ascorbate peroxidases cannot be ruled out. Mitochondrial membranes of T. cruzi epimastigotes supplemented with NADH are efficient reductants of oxygen to superoxide anion [21,22]. The membranes of endoplasmic reticulum supplemented with NADPH are also sources of superoxide anion [21]; however, these latter membranes are in an almost negligible proportion in the cell as compared to mitochondriai membranes [36]. Both activities are also responsible for the enzymatic reduction of quinones and nitroderivatives which in turn generate more superoxide anion by redox cycling [21-23]. 13-Lapachone, the o-naphthoquinone, and nifurtimox, the nitrofurane, were effective in increasing superoxide radical production (Table II), hydrogen peroxide steady state level (Table III), KCN-insensitive oxygen consumption (Fig. 6) and malondialdehyde accumulation (Fig. 7) and also markedly increased chemiluminescence (Fig. 5). Alternatively, the nitroderivative benznidazol, which does not seem to be capable of redox cycling in T. cruzi homogenates or cells [24], had no effect on the above-mentioned parameters. However, it was reported that benznidazol stimulated superoxide anion production with rat liver microsomes [24]. Chemiluminescence appears as an alternative and sensitive method for following changes in the

250 intracellular steady state concentration of oxygen r a d i c a l s in u n i c e l l u l a r e u k a r y o t e s as was p r e v i o u s l y r e p o r t e d for A . castellanii [35]. D r u g s capable of inhibiting oxygen radical catabolism or i n c r e a s i n g o x y g e n r a d i c a l p r o d u c t i o n a r e a b l e to i n c r e a s e low l e v e l c h e m i l u m i n e s c e n c e .

Acknowledgements T h e a u t h o r s wish to t h a n k D r . S t e l l a M a r l s G o n z a l e z C a p p a a n d D r . E s t e l a L a m e s for supplying Trypanosoma cruzi e p i m a s t i g o t e s that w e r e u s e d in t h e e x p e r i m e n t s . T h i s w o r k w a s supp o r t e d by t h e C o n s e j o N a c i o n a l d e I n v e s t i g a ciones Cientificas y Tecnicas (CONICET). J.F.T. a n d A . B . a r e C a r e e r I n v e s t i g a t o r s a n d C . G . is a Research Fellow from the same Institution.

References 1 Docampo, R. and Moreno, S.N.J. (1984) Free radical metabolites in the mode of action of chemotherapeutic agents and phagocytic cells on Trypanosorna cruzi. Rev. Infect. Dis. 6. 223-238. 2 Docampo, R. and Moreno, S.N.J. (1984) Free radical intermediates in the antiparasitic action of drugs and phagocytic cells. In: Free Radicals in Biology (Pryor, W.A., cd.) vol. VI, pp. 243-286, Academic Press, London. 3 Boveris, A., Sies, H., Martino, E.E., Docampo, R., Turrens, J.F. and Stoppani, A.O.M. (1980) Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem. J. 188,643--648. 4 Boveris, A. and Stoppani, A.O.M. (1977) Hydrogen peroxide generation in Trypanosorna cruzi. Experientia 33, 1306-1308. 5 Fairlamb, A.H., Blackburn, P., Ulrich, P., Chait, B.T. and Cerami, A. (1985) Trypanothione: A novel his (glutathionyl) spermidine cofactor for glutathione reductase in Trypanosomatids. Science 227, 1485-1487. 6 Shames, S., Fairlamb, A.H., Cerami, A. and Walsh, C. (1986) Puritication and characterization of trypanothione rcductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases. Biochemistry 25,351%3525. 7 Henderson, G.B., Fairlamb, A.H. and Cerami, A. (1987) Trypanothione dependent peroxide metabolism in Crithidia fasciculata and Trypanosoma brucei. Mol. Biochem. Parasitol. 24, 39-45. 8 Pcnketh, P.G. and Klein, R.A. (1986) Hydrogen peroxide metabolism in Trypanosoma brucei. Mol. Biochem. Parasitol. 20, 111-121. 9 Cadenas, E.. Boveris, A. and Chance, E. (1984) Low level chemiluminescence of biological systems. In: Free Radicals in Biology (Pryor, W.A., ed.) vol. 6, pp. 211-242. Academic Press, New York. 10 Docampo, R., de Boiso, J.F. and Stoppani, A.O.M. (1974) Diferencias metabolicas en Trypanosoma cruzi dependientes de las condiciones de cultivo. Rev. Argent. Microbiol. 6, 7-14. 11 Boveris, A., Fraga, C., Varsavsky, A.I. and Koch, O.R. (1983) Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats. Arch. Biochem. Biophys. 227, 534--541.

12 Puntarulo, S.. Sanchez, R.A. and Boveris, A. (1988) Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination. Plant Physiol. 86,626--630. 13 Boveris, A. (1984) Determination of the production of supcroxide radicals and hydrogen peroxide in mitochondria. In: Methods in Enzymology (Packer, L., ed.) vol. 1(15, pp. 429-435. Academic Press, London. 14 Misra, H.P. and Fridovich, I. (1972) The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170-3175. 15 Mc Cord, J.M. and Fridovich, I. (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049-6055. 16 Hissin, P.J. and Hill, R. (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214--226. 17 Tietze, F. (1969) Enzymatic method for quantitative determination of nanograms amounts of total and oxidized glutathione. Anal. Biochem. 27, 502-522. 18 Bernheim, F., Bernheim, M.L.C. and Wilbur, K.M. (1948) The reaction between thiobarbituric acid and the oxidation products of certain lipids. J. Biol. Chem. 174, 257-264. 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275. 20 Krauth-Siegel, R.L., Enders, B., Henderson, G.B., Fairlamb, A.tl. and Schirmer, R.H. (1987) Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme. Eur. J. Biochem. 164, 123-128. 21 Boveris, A., Docampo, R., Turrens. J.F. and Stoppani, A.O.M. (1978) Effect of 13-Lapachone on superoxide anion and hydrogen peroxide production in Trypanosoma cruzi. Biochem. J. 175,431-439. 22 Docampo, R. and Stoppani, A.O.M. (1979) Generation of superoxide anion and hydrogen peroxide induced by nifurtimox in Trypanosoma cruzi. Arch. Biochem. Biophys. 197. 317-321. 23 Docaml~), R., Moreno, S.N.J., Stoppani, A.O.M., Leon. W., Cruz, F.S., Villalta. F. and Muniz, R. (1981) Mechanism of nifurtimox toxicity in different forms of Trypanosoma cruzi. Biochem. Pharmacol. 30, 1947-1951.

251 24 Moreno, S.N.J., Docampo, R., Mason, R.P., Leon, W. and Stoppani, A.O.M. (1982) Different behaviors of benznidazole as free radical generator with mammalian and Trypanosoma cruzi microsomal preparations. Arch. Biochem. Biophys. 218, 585-591. 25 Turrens, J.F., Giulivi, C. and Boveris, A. (1986) Increased spontaneous chemiluminescence from liver homogenates and isolated hepatocytes upon inhibition of superoxide and hydrogen peroxide utilization. J. Free Rad. Biol. Med. 2, 135-140. 26 Meshnick, S., Chang, K.P. and Cerami, A. (1977) Heme lysis of the bloodstream forms of Trypanosoma brucei. Biochem. Pharmacol. 26, 1923-1928. 27 Meshnick, S.R., Blobstein, S.H., Grady, R.W. and Cerami, A. (1978) An approach to the development of new drugs for African Trypanosomiasis. J. Exp. Med. 148, 569-579. 28 Turrens, J.F., Bickar, D. and Lehninger, A.L. (1986) Inhibitors of the mitoehondrial cytochrome b-c~ complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei. Mol. Biochem. Parasitol. 19, 259-264. 29 Boveris, A., Cadenas, E. and Stoppani, A.O.M. (1976) Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J. 156,435--444.

3(I Turrens, J.F. and Boveris, A. (1980) Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191,421-427. 31 Beauchamp, C.O. and Fridovich, I. (1973) Isozymes of superoxide dismutase from wheat germ. Biochim. Biophys. Acta 317, 50-64. 32 Meshnick, S.R., I'rang, N.L., Kitchener, E., Cerami, A. and Eaton, J. (1983) Iron-containing superoxidc dismurases in Trypanosomatids. In: Oxy Radicals and their Scavenger Systems: Molecular Aspects (Cohen, G. and Greenwald, R.A., eds.) vol. 1, pp. 348-351, Elsevier-North Holland, New York. 33 Beauchamp, C. and Fridovich, 1. (197(I) A mechanism for the production of ethylene from methional. J. Biol. Chem. 245, 4641-4646. 34 Mc Cord, J.M. and Day, D.D. Jr. (19781 Superoxide dependent production of hydroxyl radical catalyzed by ironEDTA complex. FEBS Lett. 86, 139-142. 35 Lloyd, D., Boveris, A., Reiter, R., Filipkowski, M. and Chance, B. (1979) Chemiluminescence of Acanthamoeba castellanii. Biochem. J. 184, 149--156. 36 Boveris, A., Hertig, C.M. and Turrens, J.F. (1986) Fumarate reductase and other mitochondrial activities in Trypanosorna cruzi. Mol. Biochem. Parasitol. 19, 163-169.