Trypanothione-dependent peroxide metabolism in Trypanosoma cruzi different stages

Molecular and Biochemical Parasitology, 61 (1993) 79-86 © 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00

79

MOLBIO 02036

Trypanothione-dependent peroxide metabolism in Trypanosoma cruzi different stages Eva G.S. Carnieri*, Silvia N.J. M o r e n o a n d R o b e r t o D o c a m p o Department of Veterinary Pathobiology, University of Illinois, Urbana, IL, USA (Received 27 April 1993; accepted 7 June 1993)

Different stages of Trypanosoma cruzi are able to metabolize low concentrations of H202. Trypomastigotes showed a higher initial rate per mg protein than amastigotes or epimastigotes derived from them. Amastigotes could metabolize H202 at a lower rate than the other developmental stages of T. cruzi. A peroxide-metabolizing activity was detected in extracts of T. cruzi epimastigotes. This ' N A D P H peroxidase' activity was lost upon dialysis of the extracts and was probably due to a nonenzymatic reaction(s) with endogenous dihydrotrypanothione (T(SH)2) and/or other thiols, thus explaining the inhibition of H202 metabolism in intact cells by thiol inhibitors. An amount of non-protein thiols equivalent to an intracellular concentration of 2.0-3.0 mM was found in epimastigotes, which is sufficient to account for the rate of NADPH oxidation observed in the presence of high concentration of peroxides (> 100/~M). Addition of T(SH)2 increased this rate, implying that this thiol could be used as a substrate in that reaction. In addition, this activity was hardly detectable in the extracts in the presence of low concentration of peroxides (<20 /IM), indicating a high Km, which would be incompatible with a true peroxidase activity. Taking into account the high intracellular concentration of thiols measured, this activity probably accounted for the rates of H202 metabolism detected in intact 7". cruzi. These results also confirm that T. cruzi is an organism with limited ability to detoxify H202. Key words: Hydrogen peroxide; Tert-butylhydroperoxide; Trypomastigote; Amastigote; Trypanothione; Trypanosoma cruzi

Introduction

It has been reported that Trypanosoma cruzi lacks or is extremely deficient in enzyme systems necessary for the catabolism of H202 [1]. Although superoxide dismutases are present in epimastigotes [1], catalase and glutathione peroxidase are absent in these cells. Nothing is known about the presence of any of Correspondence address: Roberto Docampo, Department of Veterinary Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61801, USA. Tel.: (217)333-3845; Fax: (217)333-4628. *Permanent address: Departamento de Bioquimica, Universidade Federal de Paranfi, Curitiba, Paranfi, Brazil. Abbreviations: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid).

these enzymes in the other developmental stages of these parasites, the amastigotes and trypomastigotes. These apparent enzymatic deficiencies have been correlated with the sensitivity of T. cruzi to both intracellularly generated and phagocyte-derived reactive oxygen species [1]. The chemotherapeutic potential of these enzyme deficiencies was first recognized during the work on the mode of action of the trypanocidal o-naphthoquinone fl-lapachone and its derivatives [2]. These studies showed that the metabolism of these compounds by the parasite involved, at least in part, the generation of superoxide anion (O2-), and hence - via superoxide dismutase - o f H 2 0 2 , which accumulated in the cells to cytotoxic levels and was also excreted [2]. Unfortunately, these compounds are inactive in experimental animal infections though one

80

of them might be useful for sterilization of blood [2]. Nevertheless, the chemotherapeutic implications of these deficiencies are clear in the case of nifurtimox. Nifurtimox is a 5nitrofuran used in the treatment of Chagas' disease in humans. It seems likely that some of the cytotoxic effects of nifurtimox in T. cruzi are mediated by the generation of reactive oxygen species [3]. The evidence supporting an oxidative damage of T. cruzi induced by nifurtimox has been extensively reviewed elsewhere [1-3]. In recent years, a glutathione-spermidine cofactor termed trypanothione has been described in trypanosomatids [4]. Trypanothione appears to be essential in these organisms for maintenance of intracellular thiol states and in defense against oxidative damage [4]. A system analogous to the host glutathione reductase/ glutathione peroxidase system utilizing trypanothione has been anticipated [4]. However, most of the studies reported to date on this subject (reviewed in ref. 4), with the exception of those related to the purification [5,6], and cloning [7] of trypanothione reductase from T. cruzi, have been done using either Crithidia fasciculata, Trypanosoma brucei, or Trypanosoma congolense [4]. In additon, important gaps in our knowledge of the enzymes involved in trypanothione metabolism still exist. For example, although some authors have postulated that a trypanothione peroxidase activity occurs in T. brucei [8], another report indicated the absence of this enzyme in the same parasite [9]. The presence of such an activity in T. cruzi has not yet been investigated. Since the trypanothione system represents a particularly important target for further chemotherapeutic development, we have begun the characterization of this metabolic pathway in different stages of T. cruzi. We report here that different stages of T. cruzi are able to metabolize low levels of hydrogen peroxide, and that this activity could be attributed to non-enzymatic reactions with intracellular thiols.

Materials and Methods

Culture methods. T. cruzi trypomastigotes and amastigotes (Y strain) were obtained from the culture medium of L6E9 myoblasts by a modification of the method of Schmatz and Murray [10] as we have described before [11,12]. The final concentration of trypomastigotes and amastigotes was determined using a Neubauer chamber. The contamination of trypomastigotes with amastigotes and intermediate forms or of amastigotes with trypomastigotes or intermediate forms was always less than 5%. T. cruzi epimastigotes (Y strain) were grown at 28°C in a liquid medium consisting of brain-heart infusion (37 g 1 ~), hemin chlorohydrate (20 mg 1 1 dissolved in 50% triethanolamine), and 5% heat-inactivated newborn calf serum. Five days after inoculation, cells were collected by centrifugation. All the cells were washed twice with incubation buffer (IB) containing 5 mM KCI/ 80 mM NaCI/ 2 mM MgC12/ 16.2 mM Na2HPO4/ 3.8 mM NaH2PO4/ 50 mM glucose, adjusted to pH 7.4 at 25°C and 0.15% (w/' v) bovine serum albumin or with Dulbecco's phosphate buffered saline. The protein concentration was determined by the biuret assay [13] in the presence of 0.2% deoxycholate. L6E 9 myoblasts were cultured as described before [14]. Chemicals. N A D P H , hydrogen peroxide, tbutylhydroperoxide, fetal and newborn calf serum, and Dulbecco's phosphate buffered saline were purchased from Sigma Chemical Co. N1,NS-bis(glutathionyl)-spermidine disulfide (trypanothione, TS2) was a gift from A. Cerami or was purchased from Bachem Bioscience Inc., Philadelphia. Dihydro-trypanothione (T(SH)2) was chemically synthesized as described previously [15]. Pure recombinant trypanothione reductase was obtained as described before [7]. All other reagents were analytical grade.

H202 metabolism by intact cells'. This was measured using a slight modification of the method described by Penketh and Klein [9].

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Cells were washed with IB and suspended at a concentration of 0.1 mg protein m l - ] in IB containing 50 mg 1-1 phenol red and were incubated at 30°C. H202 was added at the start of the incubation to give a final concentration of 20/~M. Aliquots of 1.2 ml were taken every 2 or 5 min, added to micro-centrifuge tubes containing 10 ~1 horseradish peroxidase (HRP) (12 mg m l - ) , shaken, and centrifuged at 10 000 rev. rain-1 for 2 min in an Eppendorf 5415 micro-centrifuge. 20 #1 of 2 N NaOH were added per ml of supernatant and the absorbance measured at 610 nm to determine the H202 remaining. A calibration curve was constructed of absorbance against hydrogen peroxide concentration in IB for 1-20 pM H202. Solutions of H202 were prepared daily from serial dilutions of a known concentration assuming an extinction coefficient of 81 M cm-~ at 230 nm [9].

Enzyme extracts. The washed organisms were washed with Dulbecco's phosphate buffered saline, resuspended at a concentration of 2 × 10 9 cells m1-1 in a buffer containing 40 m M Hepes, pH 7.5/ 1 mM EDTA, and frozen at - 7 0 ° C . After thawing, the cells were homogenized with a Dounce homogenizer (AA, Thomas Scientific, Swedesb o r o , N J ) , and centrifuged at 10 000 rev. min -1 for 5 min in a IEC Centra MP4R micro-centrifuge. The resulting supernatants were carefully separated and used. For some experiments the supernatants were dialyzed against the same buffer (3 x 100 vol.) before analysis. For the N A D P H oxidase activity the assay medium contained 40 m M Hepes, pH 7.5, 1 m M EDTA, and 100/zM N A D P H and 0.2-0.5 mg protein in a final volume of 3 ml. Absorbance was monitored at 340 nm with reference at 430 nm using an SLM-Aminco DW2000 dual wavelength spetrophotometer. The extinction coefficient used for N A D P H was 6.22 × 103 M - 1 cm -1. For the N A D P H peroxidase activity, different concentrations of either H202 or t-butylhydroperoxide were added as indicated in the legend to Fig. 3. Trypanothione peroxidase was measured by coupling it to N A D P H oxidation which was

measured spectrophotometrically at 340-430 nm and at 30°C as indicated in the legend to Fig. 3.

Determination of non-protein thiol groups. These were measured in the homogenates by the method of Ellman [16] as modified by Sedlack and Lindsay [17]. Homogenates were prepared by disrupting the cells suspended in 40 m M Hepes pH 7.5/ l m M E D T A at a concentration of 2 x 109 cells m1-1, by freezing at - 7 0 ° C and thawing. The cells were then homogenized with a Dounce homogenizer (AA, Thomas Scientific, Swedesboro, N J) and resuspended to a concentration of 10 mg protein m l - ] in the same buffer. Results

The results shown in Fig. 1 indicate that Trypanosoma cruzi epimastigotes were able to metabolize H202 even in the absence of added glucose. This is in agreement with the well known presence of a considerable amount of endogenous substrates in the epimastigotes [18]. Heat inactivation at 100°C for 15 min led to a total inhibition of this activity (Fig. 1, HI).

HI 20

Q t_.

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= 0

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0

10

20

30

T i m e (min)

Fig. 1. Metabolism of H202 by fresh T. cruzi epimastigote suspensions. Epimastigotes were incubated at 30°C in IB containing 50 mg 1-i phenol red, with (+ Glu) or without ( - G l u ) 50 mM glucose. H202 at 20/~M final concentration was added ( t = 0 min) and aliquots were withdrawn at 2- or 5-min intervals and assayed for H202 as described under Materials and Methods. HI, the cells were heat-inactivated at 100°C for 15 min before use.

82 [

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Fig. 2. Effect of inhibitors on H202 metabolism. (A) Effect of 1 mM NaN3 (AZ); and 1 mM aminotriazole (AT) on H2O 2 metabolism by epimastigotes. To examine the effect of AT, the cells were preincubated with it for 1 h at room temperature. The corresponding controls were also preincubated for 1 h at the same temperature and their H2O 2 metabolism did not differ from that of non-preincubated cells, in these experiments, 300 #g horseradish peroxidase (HRP) m l - l was used instead of the usual 50 #g ml i in the H202 assay, due to possible inhibitory actions of these compounds on HRP. In spite of this elevated enzyme level, the sensitivity of the assay was not affected. (C)) control; (O) + NaN3 (AZ), ( A ) + aminotriazole (AT). (B) Effect of thiol reagents on H202 metabolism by epimastigotes. The cells were preincubated at room temperature with either diethylmaleate (DEM) (0.5 mg ml J, 60 min) or Nethylmaleimide (NEM) (25 #g ml i, 15 min) and then resuspended in lB. (O) control; (z~) DEM treated; (11) NEM treated; (C) Effect of 1 mM NaN3 (AZ) and 25 fig ml 1 N-ethylmaleimide (NEM) on H202 metabolism by trypomastigotes (D) Effect of I mM NaN3 (AZ) and 25 #g m l - N-ethylmaleimide (NEM) on H202 metabolism by amastigotes.

Fig. 2 shows the effect of different inhibitors on the ability of T. cruzi different stages to metabolize H202. The inhibitors of hemoprotein peroxidases aminotriazole (1 mM) and azide (1 raM), only slightly inhibited H 2 0 2 metabolism in epimastigotes (Fig. 2A). This is in agreement with the reported absence of

catalase or other hemoprotein peroxidases in epimastigotes [1]. In contrast, pretreatment with the thiol reagents N-ethylmaleimide (25 #g ml l), or diethylmaleate (0.5 mg ml-1) led to a strong inhibition of H202 metabolism (Fig. 2B). Similar results could be obtained using trypomastigotes (Fig. 2C) or amastigotes (Fig. 2D). Both T. cruzi developmental stages were able to metabolize H202 under similar conditions to those used with epimastigotes, and while azide (1 mM) had no significant effect on their ability to metabolize H202, pretreatment with N-ethylmaleimide strongly inhibited this activity. Table I shows the initial rate of H202 metabolism by T. cruzi different stages. It is interesting to note that there was a significant difference in the ability of different culture epimastigotes to metabolize H202. Epimastigotes maintained in culture for several years showed a higher initial rate of H 2 0 2 metabolism than epimastigotes of the same strain obtained by differentiation from tissue culture trypomastigotes after 5-8 passages in culture medium. Trypomastigotes showed a higher initial rate per mg protein than amastigotes TABLE 1 Initial rate of H2O 2 metabolism by different Trypanosoma cruzi stages a Stage

H202 consumption (nmol H202 m i n ] (mg protein i)

Epimastigote b Epimastigote c Amastigote Trypomastigotes

12.90 5.90 3.30 9.70

_+ 1.20 +_ 1.20 + 0.75 ± 1.30

(5) (3) (3) (3)

~Freshly washed cells (0.1 mg protein m l - i ) were incubated at 30°C in 20 ml of medium containing 5 mM KCI/80 mM N a C I / 2 m M MGC12/16.2 m M Na2HPO4/3.8 m M NaH2PO4/pH 7.4150 mM glucose/0.15% bovine serum albumin/50 mg l phenol red. H202 was added to give a final concentration of 20 /tM and 1.2-ml samples were removed at l-min intervals and assayed for residual H202 as described under Materials and Methods. H202 consumption corresponds to the first minute of exposure to 20 M H20> The values in parentheses indicate the number of experiments performed. bEpimastigotes from the Y strain maintained in culture for three years. CEpimastigotes obtained by differentiation from tissue culture trypomastigotes, after 5 8 passages in culture medium.

83 NADPH

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Fig. 3. Peroxide-dependent NADPH oxidation by T. cruzi epimastigote extracts. Control extracts (E, 0.26 mg protein ml- i) or dialyzed extracts (DE, 0.26 mg protein m1-7) were added to a medium containing 40 mM Hepes, pH 7.5/ 1 mM EDTA/0.1 mM NADPH. NADPH consumption was measured by the decrease in absorbance at 340-430 nm. Several additions of 100/~M H202 (A, C) or 100/~M t-butyl hydroperoxide (BtOOH, B, D) were made where indicated by the arrows. No changes were observed in the absence of extracts, or NADPH. The values near the tracings indicate the rate of NADPH oxidation, in nmol NADPH min 1 (mg protein)- I.

or epimastigotes derived from them. Fig. 3 shows that extracts prepared from epimastigotes as described under Materials and Methods were able to oxidize NADPH, this reaction being attributed to an NADPH oxidase, since it did not occur under N2 [19]. This NADPH oxidation was increased in a concentration-dependent manner by high concentrations (> 100 /~M) of either I"~202 o r tbutylhydroperoxide (Fig. 3A, and B) but was undetectable in the presence of low concentrations ( < 20 ~M) of peroxides. This activity, but not the NADPH oxidase activity, was lost upon dialysis of these extracts (Fig. 3C and D). Both activities (NADPH oxidase and 'NADPH peroxidase') were lost by heating the extracts at 100°C for 5 rain (not shown). Fig. 4 shows attempts to demonstrate the presence ofa trypanothione peroxidase activity in extracts of epimastigotes. Fig. 4A, trace a

--<

\

Fig. 4. Trypanothione peroxidase determination in T. cruzi epimastigote extracts. The incubation medium contained 40 mM Hepes, pH 7.5/1 mM EDTA/trypanothione reductase (0.34 U ml-1)/ 100 #M NADPH. NADPH consumption was measured by the decrease in absorbance at 340-430 nm. 100 ~M of t-butyl hydroperoxide (BtOOH) or 100/~M H202 were added where indicated. Extract (E, 1.8 mg protein ml-= (A-D)) was added where indicated by the arrows. The values near the tracings in traces b of A and C and in B and D, indicate the rate of NADPH oxidation in nmo! NADPtt m i n - i (rag protein) -I. The blank reaction due to the non-enzymatic reaction of T(SH)2 (100 ~M) and either H202 or t-butyl hydroperoxide was substracted to calculate the reaction rate in the presence of extract (A and C, traces b).

shows that addition of t-butyl hydroperoxide (I00 #M) to a suspension containing NADPH (100 /zM), trypanothione reductase (0.34 U ml - l ) and dihydrotrypanothione (100 pM, T(SH)2) slightly increased the rate of NADPH oxidation. In contrast, additon of H202 (100 #M) significantly increased this activity due to the non-enzymatic reaction between T(SH)2 and H202 (Fig. 4C, trace a). When the extract was added after NADPH alone (not shown, see Fig. 3) or in the presence of NADPH, trypanothione reductase and T(SH)2 (Fig. 4A, and C, traces'b) a similar rate of NADPH oxidation was recorded due to the NADPH oxidase activity of the preparation, in agreement with the results shown in Fig. 3. If t-butyl hydroperoxide (Fig. 4A, trace b) o r H 2 0 2 (Fig. 4C, trace b) were added after the extract, an

84

increase in the rate of N A D P H oxidation was recorded. When the peroxide was added to the extract in the absence of T(SH)2 (Fig. 4B, and D), there was an apparently lower stimulation in the rate of N A D P H oxidation ( ' N A D P H peroxidase' activity, see Fig. 3), and this rate was higher than that due to the N A D P H oxidase alone. However, discounting the blank reaction (non-enzymatic reaction of T(SH)2 with either t-butyl hydroperoxide (Fig. 4A, trace a) or H202 (Fig. 4C, trace a) and the ' N A D P H peroxidase' activity, the 'trypanothione peroxidase' activity was negligible These results are for this particular experiment but are representative of 3 experiments. Fig. 5 shows an attempt to demonstrate the presence of a trypanothione peroxidase in dialyzed epimastigote extracts with either tbutyl hydroperoxide o r H202 as substrate. In the first set of experiments (Fig. 5A) the extract was added after N A D P H . N A D P H was oxidized, this reaction being attributed to the N A D P H oxidase (see Fig. 3). Addition of trypanothione (TS2) led to an increase in N A D P H oxidation due to the trypanothione reductase activity of the extract until all the TS2 added was reduced and the rate of N A D P H oxidation returned to the original value due to the oxidase activity. Addition of

2

~rni~

o B t OOH

Bt OOH

AA: 0 . 0 8

HJO~(7.4)

Fig. 5. Assay of trypanothione peroxidase activity in T. cruzi epimastigote dialyzed extracts. The reaction mixture

(2.5 ml) contained 40 mM Hepes buffer, pH 7.5/ 1 mM EDTA/ 100 #M NADPH and the indicated additions: dialyzed extract (DE, 0.1 mg protein ml-a), trypanothione (TS2, 30 /xM), t-butyl hydroperoxide (BtOOH, 200 #M), H207 (200 /~M), TR, trypanothione reductase (0.34 U ml '). Other conditions were as described under Materials and Methods. The values near the tracings indicate the rate of NADPH oxidation, in nmol NADPH min - I (rag protein) -~. In parentheses, the rate of NADPH oxidation, in nmol NADPH m i n - ' .

either t-butyl hydroperoxide (Fig. 5A, trace a, dashed line) or H202 (Fig. 5A, trace b) further increased the rate of the overall reaction. In the second set of experiments (Figs. 5B), the same amount of TS2 was added to a reaction medium containing N A D P H and pure trypanothione reductase. After reduction of all the TS2 added, addition of either t-butyl hydroperoxide (Fig. 5B, trace a) o r H 2 0 2 (Fig. 5B, trace b) resulted in an increase in N A D P H oxidation due to the non-enzymatic reaction between T(SH)2 and either t-butyl hydroperoxide o r H 2 0 2 . It is remarkable that, in all the selected experimental conditions, the final rate of N A D P H oxidation in the presence of peroxides by the dialyzed extracts was relatively small and hardly exceeded the rate of the blank reaction mixtures from which the epimastigote extract was omitted (Fig. 5B) plus the rate of the N A D P H oxidase activity (Fig. 5A). In addition, no increase in the overall reaction (discounting both blank reactions) was observed increasing the protein concentration of the extract up to 4-fold (not shown). The increase of apparent peroxidase activity induced by the increasing concentrations of dialyzed extracts was accounted for by the N A D P H oxidase activity of the extracts (not shown). To further demonstrate the absence of a trypanothione peroxidase activity in T. cruzi epimastigotes we measured the dissapearance of T(SH)2 after incubation of the extracts in the presence of peroxides. We adapted the method of Ellman [16] who reported that 5,5'dithiobis-(2-nitrobenzoic acid) (DTNB) is reduced by SH groups to form 1 tool of 2nitro-5-mercaptobenzoic acid per mol of SH. The incubation system contained 40 m M Hepes, pH 7.5/ l m M EDTA/ 100 /~M T(SH)2/ extracts (1 mg protein ml i)/ 100 #M H202 or 100/tM t-butyl hydroperoxide in a final volume of 1 ml. Controls without extracts and without extracts and peroxides were also done. After incubation for 10 min at 30°C, 1 ml 10% trichloroacetic acid was added. The tubes were shaken intermitently for 15 min and centrifuged for 10 min at 10 000 rev. rain-~ in a IEC Centra micro-centrifuge. 2 ml

85 of the supernatant was mixed with 4.0 ml of 0.4 M Tris buffer pH 8.9/0.1 ml 0.01 M DTNB added, and the sample shaken. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no supernatant. No peroxidase activity could be detected in the extracts examined and the decrease in absorbance observed after the incubation period could be attributed entirely to the non-enzymatic reaction of T(SH)2 and either H202 or t-butyl hydroperoxide (not shown). Since no trypanothione peroxidase activity could be detected, we investigated the presence of trypanothione and other thiol compounds in T. cruzi epimastigotes. Non-protein thiol groups were measured in the extracts by the method of Ellman [16] as modified by Sedlack and Lindsay [17]. Extracts were prepared as described before. Measurement of the nonprotein thiol content in epimastigotes extracts yielded a value (mean_S.E.M.) of 6.0+2.0 nmol reduced thiols per 108 cells. Taking into account a cell volume of 30 #1 per 109 cells [20], this value represents an intracellular concentration of non-protein thiols of about 2.0-3.0 mM.

Discussion T. cruzi different stages are able to metabolize low levels of H202. The insensitivity of H202 metabolism to NaN3 and aminotriazole implies that typical (hemoprotein) catalases and peroxidases are not important in the metabolism of H202, thus confirming previous reports [1,2]. The sensitivity to thiol reagents indicates that H202 metabolism involves an essential thiol group(s). Although T. cruzi trypomastigotes showed a higher capacity to metabolize H202 as compared to epimastigotes derived from them in culture, the difference was not as high as that reported by Penketh et al. [21]. Amastigotes could metabolize H202 at a lower rate than the other developmental stages of T. cruzi. One peroxide-metabolizing activity was detected in extracts of T. cruzi epimastigotes:

a 'NADPH peroxidase'. This activity was lost upon dialysis of the extracts. One possibility is that this activity is due to a non-enzymatic reaction(s), and that the extracts contain enough amount of endogenous T(SH)2 and/ or other thiols that could be used nonenzymatically to reduce the peroxides. In this regard, an amount of non-protein thiols equivalent to an intracellular concentration of 2.0-3.0 mM was found in epimastigotes. About 36% of these non-protein thiols correspond to T(SH)2 (Docampo and Moreno, unpublished results). This represents a final concentration of about 2.8-4.2 pM endogenous thiols present in the cuvette in the experiments shown in Fig. 3A and B, which is sufficient to account for the rate of NADPH oxidation observed in the presence of high concentration of peroxides (> 100 #M). Addition of T(SH)2 increased this rate (Fig. 4A and C) therefore implying that this thiol could be used as a substrate in that reaction. The lost of this activity upon dialysis of the extracts supports the idea that it is due to a nonenzymatic reaction. Furthermore, this activity was hardly detectable in the extracts in the presence of low concentration of peroxides ( < 2 0 pM) thus indicating a high Km, which would be incompatible with a true peroxidase activity. Taking into account the high intracellular concentration of thiols measured, this activity probably accounted for the rates of H202 metabolism detected in intact cells. In conclusion, the 'peroxidase' activitiy is due to non-enzymatic reactions of endogenous reduced thiols with peroxides, thus explaining the inhibition of H202 metabolism in intact cells by thiol inhibitors. However, this activity is very low as compared to the true peroxidase activities present in mammalian cells (for example a glutathione peroxidase activity of 150 nmol m i n - l (mg protein)-1 found in lung mitochondria; ref. 1). This, together with the reported lack of catalase and glutathione peroxidase [1] confirms that T. cruzi is an organism with limited ability to detoxify H202 [1]. In other words, T. cruzi may be able to cope with a slow endogenous rate of H202 generation but it is quite sensitive to an

86

increased steady state concentration of H 2 0 2

[1]. 8

Acknowledgements 9

We thank Drs. Christopher T. Walsh and Kari Nadeau for their kind gift of recombinant trypanothione reductase and the E. coli strain containing the gene for that enzyme, and Dr. A. Cerami for his kind gift of synthetic trypanothione. This work was supported by a grant of the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases. E.G.C. is a postdoctoral fellow from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq, Brazil).

References 1 Docampo, R. (1990) Sensitivity of parasites to free radical damage by antiparasitic drugs. Chem.-Biol. Interact. 73, 1 27. 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, Vol. VI (Pryor, W., ed), pp. 243 288, Academic Press, New York. 3 Marr, J.J. and Docampo, R. (1986) Chemotherapy for Chagas' disease: a perspective of current therapy and considerations for future research. Rev. Infect. Dis. 8, 884 903. 4 Fairlamb, A.H. and Cerami, A. (1992) Metabolism and functions of trypanothione in the kinetoplastida. Annu. Rev. Microbiol. 46, 695 729. 5 Krauth-Siegel, R.L., Enders, B., Henderson, G.B., Fairlamb, A.H. and Schirmer, R.H. (1987) Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme. Eur. J. Biochem. 164, 123 128. 6 Jockers-Scheriibl, M.C., Schirmer, R.H. and KrauthSiegel, R.L. (1989) Trypanothione reductase from Trypanosoma cruzi. Catalytic properties of the enzyme and inhibition studies with trypanocidal compounds. Eur. J. Biochem. 180, 267 272. 7 Sullivan, F.X. and Walsh, C.T. (1991) Cloning,

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