ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 19’7, No. 1, October 1, pp. 317-321, 1979
Generation
of Superoxide Anion and Hydrogen Peroxide by Nifurtimox in Trypanosoma cruzil
ROBERTO Institute
DOCAMPOZ AND ANDRES
de Q&mica
0. M. STOPPAN12,3
BioQica, Facultad de Medicina, Universidad 1121 Buenos Aires, Argentina
Received
February
22, 1979; revised
Induced
de Buenos Aires,
May 8, 1979
Addition of nifurtimox (a nitrofuran derivative) to Trypanosoma cruzi culture (epimastigote) forms induced an increase in the respiratory rate and the release of H,O, from the whole cells to the suspending medium. Growth-inhibiting concentrations of nifurtimox were able to stimulate 0; production by the T. cruti mitochondrial fraction supplemented with NADH (or succinate). and also to enhance the generation of Oy by the microsomal fraction with NADPH as reductant.
Reactions [l] and [2] are believed to be the explanation of the reported “inhibition” of nitroreductase by oxygen (1, 3, 4). In the presently described study with the culture (epimastigote) form of Tqpanosoma cruxi (the agent of Chagas’ disease) we have investigated (a) factors affecting electron transfer from intracellular reducing species to a nitrofuran derivative; (b) the effect of nifurtimox4 on the production of superoxide anion and hydrogen peroxide by epimastigotes and fractions obtained therefrom, as compared with the effect on cell growth. Nifurtimox is being extensively used for the treatment of Chagas’ disease (8, 9).
Nitrofurans are known to alter the rate of cellular oxygen consumption (1, 2) and continued interest has been shown regarding their mechanism of action (3, 4). Presumably, the generation of nitrofuran radical (ArNOF) is one of the major reasons that these compounds have been effective in the treatment of anaerobic microbial infections (5). In addition, nitrofurans selectively kill anoxic mammalian cells and this anoxic toxicity is also observed with other nitroaromat.ic compounds, such as nitroimidazoles (6). Under aerobic conditions the nitrofuran radical is rapidly oxidized by molecular oxygen with regeneration of the nitro group and production of superoxide anion (1, 7). ArNOz ArNOg
enzyme
ArNO;
+ 0, -+ ArNOz + 0:
El1 Bl
1 This work was carried out with support from the Consejo National de Investigaciones Cientificas y T&znicas, the Secretaria de Estado de Ciencia y Tecnologia, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 2 Career Investigator, Consejo National de Investigaciones CientXicas y TBcnicas. 3 To whom all correspondence should be addressed at Instituto de Quimica Biologica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121Buenos Aires, Argentina.
MATERIALS
AND METHODS
Culture. T. cruzi (TulahuBn strain) was grown in Warren’s liquid medium (10) at 28°C. Six days after inoculation, cells were collected by centrifugation and washed with 0.15 M NaCl. The final concentration of epimastigotes was estimated as described before (11). Cell fractionation. To the washed epimastigote pellet glass powder (5 g/g of cells, wet wt) was added and it was ground in Q mortar for 5 min at 4°C. This procedure resulted in complete breakage of the cells, as revealed by phase contrast microscopy. The homog4 Abbreviations used: nifurtimox, 3-methyl-4-(5’-nitrofurfurylldene-amino)-tetrahydro-4H-1,4thiazine-1,1’dioxide); SOD, superoxide dismutase; HRP, horseradish peroxidase.
317
0003-9861/79/110317-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
318
DOCAMPO AND STOPPANI TABLE I
EFFECTOFNIFURTIMOXON T.cruzi Nifurtimox (rnM) 0 0.01 0.1 1.0
RESULTS RESPIRATION'
Oxygen consumption (nmol O.Jmin/mg protein) 3.9 4.9 5.2 9.2
* ” k 2
0.9b 0.8 (0.2 > P > O.l)C 0.5 (0.1 > P > 0.01) 3.4 (P < 0.001)
0 The incubation mixture contained 35 mMTris-HCl buffer (pH 7.2), 5 mM phosphate buffer (pH 7.2), 5 mM NaCl, 0.1 mM KCl, 1 mM KCN, and epimastigotes (4.0 mg protein/ml). Nifurtimox was added at the concentrations stated above. * Mean 2 SD (four independent determinations). c Significance of the difference between the nifurtimox-treated and the control samples, in terms of the t test. enate was suspended in 250 mM sucrose-5 mM KC1 (10 ml/g of cells, wet wt). Most of glass powder was separated by decantation and the suspension was subjected to differential centrifugation. The fraetions obtained were: (a) the nuclear flagellar fraction (sedimented at 680g for 10 min); (b) the mitochondrial fraction (sedimented at 30,OOOgfor 30 min); (c) the microsomal fraction (sedimented at 105,OOOgfor 60 min); (d) the supernatant. Reagents. Horseradish peroxidase (HRP), bepinephrine, erythrocyte superoxide dismutase (SOD), NADH, and NADPH were obtained from Sigma Chemical Company, St. Louis, Missouri; nifurtimox was obtained from Bayer A. G. through the courtesy of Dr. A. Haberkorn. Determination of superoxide anion and generation of hydrogen peroxide. 0; production was determined by the adrenochrome assay (12), measuring the absorption at 480-5’75 nm and using an absorption coefficient (E) of 2.96 mM-'.cm-' (13). The reaction mixture contained 1 mM epinephrine in the saline solution described before (11). H,O, generation in epimastigotes was determined by the HRP assay (14), measuring the absorption at 417-402 nm (E = 50 mM-‘.cm-I). The reaction mixtures contained 0.3-0.8 pM HRP. An Aminco-Chance double-beam spectrophotometer (American Instrument Company, Silver Springs, Maryland) was utilized. All determinations were made at 30°C. Protein was determined by the biuret method (15). Oxygen uptake. Oxygen uptake was measured in the Gilson polarograph using a platinum electrode. Assays of oxygen consumption were made in a medium containing 35 mM Tris-HCl buffer (pH 7.2), 5.0 mM phosphate buffer (pH 7.2), 5.0 mM NaCl, and 0.1 M KCl. Values in Table I (and figures) represent the average of four measurements.
As seen in Table I nifurtimox produced an initial stimulation of cyanide-insensitive respiration by T. cruxi epimastigotes, although with prolonged incubation inhibition occurred (not shown). No increase in the 0, consumption was observed upon addition of nifurtimox in the absence of epimastigotes. The stimulation of cellular oxygen utilization involved a direct drug radical reaction with oxygen (Reactions [l] and [2]>, in good agreement with the effect of nifurtimox on the rate of hydrogen peroxide production in Fig. 1. Respiring epimastigotes did not release H,O, to the suspending medium (16) but addition of nifurtimox initiated and immediate production of H,O, (Fig. 1). Without epimast.igotes present in the assay medium no increase of A41,--402occurred upon addition of nifurtimox. As in previous observations (17), the mitochondrial fraction of T. cruzi actively generated 0; in the presence of NADH (Fig. 2). Addition of nifurtimox to this fraction supplemented with NADH, stimulated 0; production almost linearly up to 20 PM nifurtimox, at which concentration the basal
,
-
N
$10 NIFURTIHOX ()IM)
FIG. 1. Effect of nifurtimox concentration on H,O, release from whole epimastigotes. The incubation mixture contained 35 mM Tris-HCl buffer (pH 7.2), 5.0 mM phosphate buffer (pH 7.2), 5.0 mM NaCl, 0.1 M KCl, 0.6 pM horseradish peroxidase, and epimastigotes (0.1-0.2 mg protein/ml). Inset: Typical tracings showing nifurtimox-induced H,Oz production by epimastigotes. E and N, epimastigote and nifurtimox additions, respectively. The numbers near the traces indicate nifurtimox concentration (PM). Other experimental conditions were as described in the text.
EFFECTS OF NIFURTIMOX
40 20 NIFURTIMOX (fib41
60
FIG. 2. Effect of nifurtimox on 05 production by the mitochondrial fraction. The incubation mixture contained 0.23 M mannitol, 0.07 M sucrose, 50 mM TrisHCI buffer (pH 7.4), 1 mM EDTA, 1 mM adrenaline, and 0.1-0.4 mg protein. Where indicated, 40 pM NADH or NADPH, or 3 mM succinate @UC).
rate was increased threefold. Lesser effects were observed with NADPH or succinate as reductant. Production of 0; by the microsomal fraction was also stimulated by nifurtimox (Fig. 3). In the presence of NADPH, nifurtimox was able to stimulate 0~ generation approximately twofold at about 40 PM. Much lesser effects were observed with NADH as electron donor. Nifurtimox-stimulated adrenochrome formation did not occur in the absence of mitochondrial or microsomal fractions with either NADH, NADPH, or succinate. Nifurtimox concentrations increasing 0% and H,Op production by epimastigotes and cell fractions were effective in inhibiting parasite growth as shown by Fig. 4.
319
IN Tryparwsowm cmzi
40
80 120 NIFURTIMOX (FM)
160
FIG. 3. Effect of nifurtimox on 0~ production by the microsomal fraction. The incubation mixture contained 0.13 M KCl, 20 mM potassium phosphate buffer (pH 7.2), 1 mM adrenaline and 0.1-0.4 mg protein. Where indicated, 40 pM NADH or NADPH. In all cases adrenochrome formation was inhibited completely by adding 5 pg SOD/ml. Other experimental conditions were as described in the text.
membrane to the extracellular medium does not seem to occur under physiological conditions (Fig. 1 and Ref. (16)). In consequence, the trypanosomes must have little or no significant H,O, production under normal conditions. Since H,O, is toxic for T. cruxi and other Trypanosomatidae (18, 19), substances like nifurtimox. that increase
DISCUSSION
The main defense mechanism of T. cruzi epimastigotes against the accumulation of the intermediates of the partial reduction of oxygen includes superoxide dismut.ase and peroxidase (16, 17). The former enzyme is present in the homogenate with an activity of 0.28 pugof bovine superoxide dismutasel mg of protein (16). The ascorbat.e-utilizing peroxidase is microperoxisomal, but it is also to be found in t.he cyt.osol to a limited extent (17). T. cruxi does not contain catalase (17) and diffusion of H,O, through cell
NIFURTIMOX
(fibI
FIG. 4. Effect of nifurtimox on growth of T. cruzi. Experimental conditions were as described in the text. The generation time (mean) is the time (T) required for the number of cells to double during the logarithmic phase. Inset: Percentage inhibition of growth calculated from the growth rate constant (k = 0.69/T) at different nifurtimox concentrations.
320
DOCAMPO
AND STOPPANI
the intracellular H,O, generation, are possible trypanocides. Addition of nifurtimox to T. cruxi epimastigotes increases the rate of oxygen consumption and of intracellular H,Oz generation (Table I and Fig. 1). The response indicates a rapid permeation of nifurtimox through the epimastigote cell membrane. The chemical reactions following the introduction of the nitrofuran derivative into epimastigotes could be written:
which is as high as that which produces maximal stimulation of Op production by the T. cruzi mitochondrial fraction (Fig. 2>, initiates diffusion of H,O, outside of the cells (Fig. 1) and completely inhibits growth (Fig. 4). It is noteworthy, that other bactericidal and cytotoxic drugs such as paraquat (28, 29) probably share with nifurtimox the same general mechanism of biological action. ACKNOWLEDGMENTS
NAD(P)H + H+ + 2ArN02 -+ 2ArNOp + NAD(P)+ + 2H+ ArNO? + 0, + ArNOz + Op
[3] [41
We are grateful to Lucrecia Bertolini de Villalba and Beatriz Seguel de Iezzi for their excellent technical assistance. REFERENCES
0~ + 0; + 2H+ %
H,O, + 0,
0% + H,O, +Fe3+ 0, + OH’ + OH-.
[5]
WI
Reaction [3] is the nitroreductase reaction (20) that takes place in the mitochondrial and endoplasmic reticulum membranes. Reaction [4] is known to occur in chemical systems (4). A rapid O$ formation was observed in the mitochondrial fraction of T. cr~zi epimastigotes in the presence of nifurtimox and NADH (Fig. 2) and the same occurred with the microsomal fraction with NADPH as electron donor (Fig. 3). Superoxide dismutation yields H,O, (Reaction [5]) (21) and Reaction [6], which is now thought to depend upon catalysis by iron compounds (22>, leads to the production of hydroxyl radical, which is known to be one of the most damaging reactive species resulting from the univalent pathway of oxygen reduction (21). Reactions [3] to [6] may lead to extensive DNA damage (23) and also to lipid and organic peroxide formation. In this connection it is worth recalling that T. cruzi epimastigotes supplemented with H,O,producing, growth-inhibiting naphthoquinones showed (19, 24, 25) increased lipid peroxidation (19), leading to enzyme inactivation and membrane damage. The ultrastructural and metabolic alterations observed in nifurtimox-treated T. cruzi cells (26) may involve the same mechanism. The serum concentration of nifurtimox after administration of a single oral dose of 15 me/kc in man is about lo-20 uM (27).
1. BIAGLOW, J. E., GREENSTOCK, C. L., JACOBSON, B., AND RALEIGH, J. (1977) Mol. Pharrnucol. 13, 269-282. 2. BIAGLOW, J. E., NYGAARD, 0. F., AND GREENSTOCK, C. L. (1976) Biochem. Pharmacol. 25, 393-398. 3. MASON, R. P., AND HOLTZMAN, J. L. (1975) Biothem. Biophys. Res. Commun. 67, 1267-1274. 4. WARDMAN, P., AND CLARKE, E. D. (1976) Biothem. Biophys. Res. Commun. 69, 942-949. 5. GRUNBERG, E., AND TITSWORTH, E. H. (1973) Annu. Rev. Microbial. 27, 317-346. 6. MOHINDRA, J. K., AND RAUTH, A. M. (1976) Cancer Res. 36, 930-936. 7. SEALEY, R. C., SWARTZ, H. M., AND OLIVE, P. L. (1978) Biochem. Biophys. Res. Commun. 82, 680-684. 8. GUTTERIDGE, W. E. (1976) in New Approaches in American Trypanosomiasis Research, pp. 255-262, PAHO Scientific Publ. N”318. 9. VAN DEN BOSSCHE, H. (1978) Nature (London) 173, 626-630. 10. WARREN, L. (1960) J. Parasitol. 46, 529-539. 11. DOCAMPO, R., BOISO, J. F. DE, AND STOPPANI, A. 0. M. (1978) Biochim. Biophys. Acta 502, 466-476. 12. MISRA, H. P., AND FRIWVICH, I. (1972) J. Biol. Chm. 247, 188-192. 13. GREEN, S., MAZUR, A., AND SHORR, E. (1956) J. Biol. Chem. 220, 237-255. 14. BOVERIS, A., MARTINO, E., AND STOPPANI, A. 0. M. (1977) Anal. Biochem. 80, 145-158. 15. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. 16. BOVERIS, A., AND STOPPANI, A. 0. M. (1977) Experientia 33, 1366- 1308. 17. DOCAMPO, R., BOISO, J. F. DE, BOVERIS, A., AND STOPPANI, A. 0. M. (1976) Experientia 32, 972-975.
EFFECTS
OF NIFURTIMOX
18. FULTON, J. D., AND SPOONER, D. F. (1956) Biochm. J. 63, 475-481. 19. DOCAMPO, R., CRUZ, F. S., BOVERIS, A., MUNIZ, R. P. A., AND ESQUIVEL, D. M. S. (1978)Arch. Biochem. Biophys. 186.292-297. 20. MASON, R. P., AND HOLTZMAN, J. L. (1975) Biochemistry 14, 1626- 1632. 21. FRIDOVICH, I. (1978) Science 201, 875-880. 22. MCCORD, J. M., AND DAY, E. D. JR. (1978)FEBS Lett. 86, 139-142. 23. OLIVE, P. L. (1978) Chm. Biol. Interact. 20,323331. 24. BOVERIS, A., DOCAMPO, R., TURRENS, J. F., AND
IN Trypanosoma
25.
26. 27.
28. 29.
cruzi
321
STOPPANI, A. 0. M. (1978) Biochem. J. 175, 431-439. BOVERIS, A., STOPPANI, A. 0. M., DOCAMPO, R., AND CRUZ, F. S. (1978) Comp. Biochem. Physiol. 61C, 327-329. VOIGT, W. H., HABERKORN, A., AND G~NNERT, R. (1973) 2. Parasitenk. 41, 255-267. MEDENWALD, H., BRANDAU, K., AND SCHLOSSMANN, K. (1972)Arzneim-Forsch. (Drugs Res.) 22, 1613-161’7. HASSAN, H. M., AND FRID~VICH, I. (1977) J. Biol. Chem. 252, 7667-7672. HASSAN, H. M., AND FRIWVICH, I. (1978) J. Biol. Chem. 253, 8143-8148.
Generation
of Superoxide Anion and Hydrogen Peroxide by Nifurtimox in Trypanosoma cruzil
ROBERTO Institute
DOCAMPOZ AND ANDRES
de Q&mica
0. M. STOPPAN12,3
BioQica, Facultad de Medicina, Universidad 1121 Buenos Aires, Argentina
Received
February
22, 1979; revised
Induced
de Buenos Aires,
May 8, 1979
Addition of nifurtimox (a nitrofuran derivative) to Trypanosoma cruzi culture (epimastigote) forms induced an increase in the respiratory rate and the release of H,O, from the whole cells to the suspending medium. Growth-inhibiting concentrations of nifurtimox were able to stimulate 0; production by the T. cruti mitochondrial fraction supplemented with NADH (or succinate). and also to enhance the generation of Oy by the microsomal fraction with NADPH as reductant.
Reactions [l] and [2] are believed to be the explanation of the reported “inhibition” of nitroreductase by oxygen (1, 3, 4). In the presently described study with the culture (epimastigote) form of Tqpanosoma cruxi (the agent of Chagas’ disease) we have investigated (a) factors affecting electron transfer from intracellular reducing species to a nitrofuran derivative; (b) the effect of nifurtimox4 on the production of superoxide anion and hydrogen peroxide by epimastigotes and fractions obtained therefrom, as compared with the effect on cell growth. Nifurtimox is being extensively used for the treatment of Chagas’ disease (8, 9).
Nitrofurans are known to alter the rate of cellular oxygen consumption (1, 2) and continued interest has been shown regarding their mechanism of action (3, 4). Presumably, the generation of nitrofuran radical (ArNOF) is one of the major reasons that these compounds have been effective in the treatment of anaerobic microbial infections (5). In addition, nitrofurans selectively kill anoxic mammalian cells and this anoxic toxicity is also observed with other nitroaromat.ic compounds, such as nitroimidazoles (6). Under aerobic conditions the nitrofuran radical is rapidly oxidized by molecular oxygen with regeneration of the nitro group and production of superoxide anion (1, 7). ArNOz ArNOg
enzyme
ArNO;
+ 0, -+ ArNOz + 0:
El1 Bl
1 This work was carried out with support from the Consejo National de Investigaciones Cientificas y T&znicas, the Secretaria de Estado de Ciencia y Tecnologia, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 2 Career Investigator, Consejo National de Investigaciones CientXicas y TBcnicas. 3 To whom all correspondence should be addressed at Instituto de Quimica Biologica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121Buenos Aires, Argentina.
MATERIALS
AND METHODS
Culture. T. cruzi (TulahuBn strain) was grown in Warren’s liquid medium (10) at 28°C. Six days after inoculation, cells were collected by centrifugation and washed with 0.15 M NaCl. The final concentration of epimastigotes was estimated as described before (11). Cell fractionation. To the washed epimastigote pellet glass powder (5 g/g of cells, wet wt) was added and it was ground in Q mortar for 5 min at 4°C. This procedure resulted in complete breakage of the cells, as revealed by phase contrast microscopy. The homog4 Abbreviations used: nifurtimox, 3-methyl-4-(5’-nitrofurfurylldene-amino)-tetrahydro-4H-1,4thiazine-1,1’dioxide); SOD, superoxide dismutase; HRP, horseradish peroxidase.
317
0003-9861/79/110317-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
318
DOCAMPO AND STOPPANI TABLE I
EFFECTOFNIFURTIMOXON T.cruzi Nifurtimox (rnM) 0 0.01 0.1 1.0
RESULTS RESPIRATION'
Oxygen consumption (nmol O.Jmin/mg protein) 3.9 4.9 5.2 9.2
* ” k 2
0.9b 0.8 (0.2 > P > O.l)C 0.5 (0.1 > P > 0.01) 3.4 (P < 0.001)
0 The incubation mixture contained 35 mMTris-HCl buffer (pH 7.2), 5 mM phosphate buffer (pH 7.2), 5 mM NaCl, 0.1 mM KCl, 1 mM KCN, and epimastigotes (4.0 mg protein/ml). Nifurtimox was added at the concentrations stated above. * Mean 2 SD (four independent determinations). c Significance of the difference between the nifurtimox-treated and the control samples, in terms of the t test. enate was suspended in 250 mM sucrose-5 mM KC1 (10 ml/g of cells, wet wt). Most of glass powder was separated by decantation and the suspension was subjected to differential centrifugation. The fraetions obtained were: (a) the nuclear flagellar fraction (sedimented at 680g for 10 min); (b) the mitochondrial fraction (sedimented at 30,OOOgfor 30 min); (c) the microsomal fraction (sedimented at 105,OOOgfor 60 min); (d) the supernatant. Reagents. Horseradish peroxidase (HRP), bepinephrine, erythrocyte superoxide dismutase (SOD), NADH, and NADPH were obtained from Sigma Chemical Company, St. Louis, Missouri; nifurtimox was obtained from Bayer A. G. through the courtesy of Dr. A. Haberkorn. Determination of superoxide anion and generation of hydrogen peroxide. 0; production was determined by the adrenochrome assay (12), measuring the absorption at 480-5’75 nm and using an absorption coefficient (E) of 2.96 mM-'.cm-' (13). The reaction mixture contained 1 mM epinephrine in the saline solution described before (11). H,O, generation in epimastigotes was determined by the HRP assay (14), measuring the absorption at 417-402 nm (E = 50 mM-‘.cm-I). The reaction mixtures contained 0.3-0.8 pM HRP. An Aminco-Chance double-beam spectrophotometer (American Instrument Company, Silver Springs, Maryland) was utilized. All determinations were made at 30°C. Protein was determined by the biuret method (15). Oxygen uptake. Oxygen uptake was measured in the Gilson polarograph using a platinum electrode. Assays of oxygen consumption were made in a medium containing 35 mM Tris-HCl buffer (pH 7.2), 5.0 mM phosphate buffer (pH 7.2), 5.0 mM NaCl, and 0.1 M KCl. Values in Table I (and figures) represent the average of four measurements.
As seen in Table I nifurtimox produced an initial stimulation of cyanide-insensitive respiration by T. cruxi epimastigotes, although with prolonged incubation inhibition occurred (not shown). No increase in the 0, consumption was observed upon addition of nifurtimox in the absence of epimastigotes. The stimulation of cellular oxygen utilization involved a direct drug radical reaction with oxygen (Reactions [l] and [2]>, in good agreement with the effect of nifurtimox on the rate of hydrogen peroxide production in Fig. 1. Respiring epimastigotes did not release H,O, to the suspending medium (16) but addition of nifurtimox initiated and immediate production of H,O, (Fig. 1). Without epimast.igotes present in the assay medium no increase of A41,--402occurred upon addition of nifurtimox. As in previous observations (17), the mitochondrial fraction of T. cruzi actively generated 0; in the presence of NADH (Fig. 2). Addition of nifurtimox to this fraction supplemented with NADH, stimulated 0; production almost linearly up to 20 PM nifurtimox, at which concentration the basal
,
-
N
$10 NIFURTIHOX ()IM)
FIG. 1. Effect of nifurtimox concentration on H,O, release from whole epimastigotes. The incubation mixture contained 35 mM Tris-HCl buffer (pH 7.2), 5.0 mM phosphate buffer (pH 7.2), 5.0 mM NaCl, 0.1 M KCl, 0.6 pM horseradish peroxidase, and epimastigotes (0.1-0.2 mg protein/ml). Inset: Typical tracings showing nifurtimox-induced H,Oz production by epimastigotes. E and N, epimastigote and nifurtimox additions, respectively. The numbers near the traces indicate nifurtimox concentration (PM). Other experimental conditions were as described in the text.
EFFECTS OF NIFURTIMOX
40 20 NIFURTIMOX (fib41
60
FIG. 2. Effect of nifurtimox on 05 production by the mitochondrial fraction. The incubation mixture contained 0.23 M mannitol, 0.07 M sucrose, 50 mM TrisHCI buffer (pH 7.4), 1 mM EDTA, 1 mM adrenaline, and 0.1-0.4 mg protein. Where indicated, 40 pM NADH or NADPH, or 3 mM succinate @UC).
rate was increased threefold. Lesser effects were observed with NADPH or succinate as reductant. Production of 0; by the microsomal fraction was also stimulated by nifurtimox (Fig. 3). In the presence of NADPH, nifurtimox was able to stimulate 0~ generation approximately twofold at about 40 PM. Much lesser effects were observed with NADH as electron donor. Nifurtimox-stimulated adrenochrome formation did not occur in the absence of mitochondrial or microsomal fractions with either NADH, NADPH, or succinate. Nifurtimox concentrations increasing 0% and H,Op production by epimastigotes and cell fractions were effective in inhibiting parasite growth as shown by Fig. 4.
319
IN Tryparwsowm cmzi
40
80 120 NIFURTIMOX (FM)
160
FIG. 3. Effect of nifurtimox on 0~ production by the microsomal fraction. The incubation mixture contained 0.13 M KCl, 20 mM potassium phosphate buffer (pH 7.2), 1 mM adrenaline and 0.1-0.4 mg protein. Where indicated, 40 pM NADH or NADPH. In all cases adrenochrome formation was inhibited completely by adding 5 pg SOD/ml. Other experimental conditions were as described in the text.
membrane to the extracellular medium does not seem to occur under physiological conditions (Fig. 1 and Ref. (16)). In consequence, the trypanosomes must have little or no significant H,O, production under normal conditions. Since H,O, is toxic for T. cruxi and other Trypanosomatidae (18, 19), substances like nifurtimox. that increase
DISCUSSION
The main defense mechanism of T. cruzi epimastigotes against the accumulation of the intermediates of the partial reduction of oxygen includes superoxide dismut.ase and peroxidase (16, 17). The former enzyme is present in the homogenate with an activity of 0.28 pugof bovine superoxide dismutasel mg of protein (16). The ascorbat.e-utilizing peroxidase is microperoxisomal, but it is also to be found in t.he cyt.osol to a limited extent (17). T. cruxi does not contain catalase (17) and diffusion of H,O, through cell
NIFURTIMOX
(fibI
FIG. 4. Effect of nifurtimox on growth of T. cruzi. Experimental conditions were as described in the text. The generation time (mean) is the time (T) required for the number of cells to double during the logarithmic phase. Inset: Percentage inhibition of growth calculated from the growth rate constant (k = 0.69/T) at different nifurtimox concentrations.
320
DOCAMPO
AND STOPPANI
the intracellular H,O, generation, are possible trypanocides. Addition of nifurtimox to T. cruxi epimastigotes increases the rate of oxygen consumption and of intracellular H,Oz generation (Table I and Fig. 1). The response indicates a rapid permeation of nifurtimox through the epimastigote cell membrane. The chemical reactions following the introduction of the nitrofuran derivative into epimastigotes could be written:
which is as high as that which produces maximal stimulation of Op production by the T. cruzi mitochondrial fraction (Fig. 2>, initiates diffusion of H,O, outside of the cells (Fig. 1) and completely inhibits growth (Fig. 4). It is noteworthy, that other bactericidal and cytotoxic drugs such as paraquat (28, 29) probably share with nifurtimox the same general mechanism of biological action. ACKNOWLEDGMENTS
NAD(P)H + H+ + 2ArN02 -+ 2ArNOp + NAD(P)+ + 2H+ ArNO? + 0, + ArNOz + Op
[3] [41
We are grateful to Lucrecia Bertolini de Villalba and Beatriz Seguel de Iezzi for their excellent technical assistance. REFERENCES
0~ + 0; + 2H+ %
H,O, + 0,
0% + H,O, +Fe3+ 0, + OH’ + OH-.
[5]
WI
Reaction [3] is the nitroreductase reaction (20) that takes place in the mitochondrial and endoplasmic reticulum membranes. Reaction [4] is known to occur in chemical systems (4). A rapid O$ formation was observed in the mitochondrial fraction of T. cr~zi epimastigotes in the presence of nifurtimox and NADH (Fig. 2) and the same occurred with the microsomal fraction with NADPH as electron donor (Fig. 3). Superoxide dismutation yields H,O, (Reaction [5]) (21) and Reaction [6], which is now thought to depend upon catalysis by iron compounds (22>, leads to the production of hydroxyl radical, which is known to be one of the most damaging reactive species resulting from the univalent pathway of oxygen reduction (21). Reactions [3] to [6] may lead to extensive DNA damage (23) and also to lipid and organic peroxide formation. In this connection it is worth recalling that T. cruzi epimastigotes supplemented with H,O,producing, growth-inhibiting naphthoquinones showed (19, 24, 25) increased lipid peroxidation (19), leading to enzyme inactivation and membrane damage. The ultrastructural and metabolic alterations observed in nifurtimox-treated T. cruzi cells (26) may involve the same mechanism. The serum concentration of nifurtimox after administration of a single oral dose of 15 me/kc in man is about lo-20 uM (27).
1. BIAGLOW, J. E., GREENSTOCK, C. L., JACOBSON, B., AND RALEIGH, J. (1977) Mol. Pharrnucol. 13, 269-282. 2. BIAGLOW, J. E., NYGAARD, 0. F., AND GREENSTOCK, C. L. (1976) Biochem. Pharmacol. 25, 393-398. 3. MASON, R. P., AND HOLTZMAN, J. L. (1975) Biothem. Biophys. Res. Commun. 67, 1267-1274. 4. WARDMAN, P., AND CLARKE, E. D. (1976) Biothem. Biophys. Res. Commun. 69, 942-949. 5. GRUNBERG, E., AND TITSWORTH, E. H. (1973) Annu. Rev. Microbial. 27, 317-346. 6. MOHINDRA, J. K., AND RAUTH, A. M. (1976) Cancer Res. 36, 930-936. 7. SEALEY, R. C., SWARTZ, H. M., AND OLIVE, P. L. (1978) Biochem. Biophys. Res. Commun. 82, 680-684. 8. GUTTERIDGE, W. E. (1976) in New Approaches in American Trypanosomiasis Research, pp. 255-262, PAHO Scientific Publ. N”318. 9. VAN DEN BOSSCHE, H. (1978) Nature (London) 173, 626-630. 10. WARREN, L. (1960) J. Parasitol. 46, 529-539. 11. DOCAMPO, R., BOISO, J. F. DE, AND STOPPANI, A. 0. M. (1978) Biochim. Biophys. Acta 502, 466-476. 12. MISRA, H. P., AND FRIWVICH, I. (1972) J. Biol. Chm. 247, 188-192. 13. GREEN, S., MAZUR, A., AND SHORR, E. (1956) J. Biol. Chem. 220, 237-255. 14. BOVERIS, A., MARTINO, E., AND STOPPANI, A. 0. M. (1977) Anal. Biochem. 80, 145-158. 15. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. 16. BOVERIS, A., AND STOPPANI, A. 0. M. (1977) Experientia 33, 1366- 1308. 17. DOCAMPO, R., BOISO, J. F. DE, BOVERIS, A., AND STOPPANI, A. 0. M. (1976) Experientia 32, 972-975.
EFFECTS
OF NIFURTIMOX
18. FULTON, J. D., AND SPOONER, D. F. (1956) Biochm. J. 63, 475-481. 19. DOCAMPO, R., CRUZ, F. S., BOVERIS, A., MUNIZ, R. P. A., AND ESQUIVEL, D. M. S. (1978)Arch. Biochem. Biophys. 186.292-297. 20. MASON, R. P., AND HOLTZMAN, J. L. (1975) Biochemistry 14, 1626- 1632. 21. FRIDOVICH, I. (1978) Science 201, 875-880. 22. MCCORD, J. M., AND DAY, E. D. JR. (1978)FEBS Lett. 86, 139-142. 23. OLIVE, P. L. (1978) Chm. Biol. Interact. 20,323331. 24. BOVERIS, A., DOCAMPO, R., TURRENS, J. F., AND
IN Trypanosoma
25.
26. 27.
28. 29.
cruzi
321
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