- Email: [email protected]
Inhibition of growth and respiration of leishmania mexicana by the antitumor agent lonidamine
Camp. Biochem. Physid.
0306~4492/87 $3.00+ 0.00 0 1987 Pergamon Journals Ltd
Vol. 88C, No. I, pp, 193-196, 1987
Printed in Great Britain
INHIBITION OF GROWTH AND RESPIRATION OF LEI,!$HMANIA MEXICANA BY THE ANTITUMOR AGENT LONIDAMINE JULIO F. TURRENS* and JUAN Jo& CAZZULO~ *Institute de Quimica y Fisicoqu~mica Biol6gicas, Facultad de Farmacia y Bioqu~mi~, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina; ?Instituto de Investigaciones Bioquimicas Fundaci6n Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina. Telephone: 88-1916 (Received 27 November 1986) Abstract-1. The antitumor drug lonidamine inhibited growth of promastigotes of Leishmunia mexicana in axenic culture. 2. Fifty percent inhibition was attained at 0.42 mM, and was reflected mainly in an increase in lag time, with less effect on final cell yield. 3. The drug was leishmanistatic, since when a non-growing culture in the presence of 0.5 mM lonidamine was centrifuged and the cells resuspended in fresh medium, growth started and reached the control value. 4. Both coupled and FCCP-uncoupled respiration of intact promastigotes were inhibited by lonidamine; 50% inhibition was attained at 0.5 and 0.4 mM, respectively. 5. The results suggested that the mechanism of inhibition of growth of L. mexicona is, as proposed in the case of Try~~nosvrn~cruzi epimastigotes and ~ryp~nosom~ brucei procyclic try~mastigotes, through inhibition of the energy metabolism.
INTRODUCTION Lonidamine (I-(2,4-dichlorobenzyl-1 ,H-indazol-3carboxylic acid) (Fig. 1) is a drug which was first
developed as an antis~~atogenic agent (Silvestrini et al., 1975; Coulston et al., 1975), and was later found effective against experimental tumors (Floridi et al., 1981; Silvestrini et al., 1984); it has relatively low toxicity, and clinical trials as an antitumor agent are under way (Band et al., 1984; Young et al., 1984). The mode of action of lonidamine on mammalian cells involves inhibition of energy metabolism at different levels, including the particle-bound hexokinase and uncoupled respiration (Floridi et al., 1981; Floridi and Lehninger, 1983). Recently, one of us has reported inhibition by lonidamine of growth and uncoupled respiration of Trypanosoma cruzi, the parasitic flagellate which causes the American trypanosomiasis, Chagas’ disease; uncoupled respiration of Trypanosoma brucei procyclic trypomastigotes was also inhibited by the drug, whereas the cyanidesalicyl hydroxamic acid (SHAM)insensitive, sensitive respiration of blood-stream trypomastigotes of T. brucei was much less affected (Turrens, 1986). In the present report we extend this study to another pathogenic trypanosomatid, Leishmu~ia mexicana. Growth in axenic culture of promastigotes and uncoupled respiration of the intact cells were inhibited by lonidamine, although the concentrations required were considerably higher than in the case of T. cruzi and T. brucei. Interestingly, the drug at 0.5 mM was only leishmanistatic, whereas the same concentration was clearly trypanocide against T.
University of Costa Rica, San Jo&, Costa Rica, and lr. cruzi, Tul 0 stock, were kindly given by Dr Elsa L. Segura, Instituto Fatala Chaben, Buenos Aires, Argentina. L. mexicana promastigotes and T. brucei epimastigotes were grown in a brain heart tryptose medium, containing 20% (L. mexicana) or 10% (T. cruzi) fetal calf serum (Cazzulo et al., 1985). Freshly inoculated culture medium was divided into IO-ml samples which were incubated at 25°C (L. mexicana) or 28°C (T. cruzi) in 125-ml Erlenmeyer flasks fitted with a lateral tube which allowed growth to be followed in a Lumetron Colorimeter (Photovolt Company) using a red filter. Since theese organisms form rosettes only in the stationary phase, turbidity was proportional to cell numbers. Cell motility and integrity was checked by microscopic observation. Lonidamine was added as a 50 mM solution in dimethyl-sulfoxide (DMSO). Appropriate controls indicated that even the highest DMSO concentration used (0. I in IOml of culture medium) caused negligible effects on growth per se. Respiration experiments For these experiments, promastigotes of L. mexicana were grown in the same culture medium in Roux flasks at 25”C, and harvested in the late exponential phase of growth (about 40 x 106cells/ml). The cells were harvested by centrifugation at 5800g for IS min at 4”C, and washed twice in
cruzi.
: OH clJT-+ ’ ,,i I
xl‘1
Cl
MATERIALS AND METHODS
Cl
Lonidamine
Organism and growth L. mexicana, originally isolated by Dr Rodrigo Zeledbn, C.B P. 88,1C--M
0
Fig. 1. Structure of lonidamine. 193
JULIOF. TURRENSand JUAN Jo& CAZZULO
194
shape of the inhibition curve for L. mexicana is bizarre, since 0.4mM lonidamine inhibited by 36% and 0.5mM inhibited by 100%. This was not an artifact caused by an adverse effect of the solvent, and probably reffects an extreme lengthening of the lag phase. In fact, the cells were still alive after 5 days incubation in the presence of 0.5 mM lonidamine, since when the culture medium containing the drug was removed by centrifugation and the cells resuspended in fresh medium, the promastigotes started growing after a lag phase of nearly 3 days and reached the same absorbance value as the control (Fig. 3). Microscopic observation showed that the cells had normal morphology and motility, either in the presence or in the absence of lonidamine. When an experiment similar to that of Fig. 3 was performed with T. cruzi, the cells incubated with 0.5 mM lonidamine, centrifuged and resuspended in fresh medium, did not resume growth, showing that the drug is trypanocide at this concentration (not shown). Figure 4 shows that lonidamine has an inhibitory effect on the respiration of intact L. mexicana cells, in the absence or in the presence of the uncoupler carbonyl - cyanide -p - trifluoromethoxyphenylhydra zone (FCCP).
the centrifuge with 250 mM sucrose-5 mM KCl. Oxygen consumption by the cells, suspended in the same medium, was monitored with a Clark electrode at 3o”C, using a medium containing 6mM glucose, 140mM NaCl and 10 mM Tris-HCI @H 7.4).
RESULTS Figure 2a, shows that lonidamine inhibited growth of L. mexicana in culture, although it was less effective than in the case of T. cruzi (Fig. 2b). There was a clear difference in the effects, since in the case of T. cruzi the cultures in the presence of the drug grew at a lower rate and reached a lower stationary phase, whereas in the case of L. mexicana the main effect was on the lag phase, which was progressively lengthened by increasing concentrations of the drug, with less effect on the cell concentration at the stationary phase. Figure 2c, shows cell growth as a function of lonidamine concentration; the data were taken from Fig. 2a and b. The concentration required for a 50% decrease in cell concentration of L. mexicana at the stationary phase (I~,,) was about 0.42 mM, whereas the value for T. cruzi in the present experimental
conditions
was
about
0.2 mM.
The r-
I
0
0.125 0.25 0.40
I
I
0
tb I
(dl
I
L
i” 0.2!5
0.54I_
>
I
L
I
100
20 0 Time
in
culture
Lonidamine
(hrl
ImMl
Fig. 2. Inhibition of growth of L. mexicana promastigotes (a) and T. cruzi epimastigotes Tul 0 stock (b) by lonidamine. Numbers on the curves indicate lonidamine concentration (mM). (c) Inhibition of growth of L. mexicana (0) and Z’. cruzi (0) ccl1 concentration at the stationary phase, as a function of drug concentration. The experimental points are the average of duplicates which differed at most by 10%.
Lonidamine inhibits Leishmunia
195
(0.2 mM). The latter value, obtained with the Tul 0 stock of T. cruzi, was 2.5-fold greater than that previously found with the Tul 2 stock of the same organism (Turrens, 1986). This difference might be due to strain differences, since marked differences in the effectiveness of the trypanocidal drugs nifurtimox, benznidazole and allopurinol, have been reported, depending on the parasite strain used (Andrade et al., 1985; Avila and Mufioz, 1981). Moreover, different batches of lonidamine were used in both sets of experiments. Differences in the effect of lonidamine on L. mexicana and T. cruzi are emphasized by the fact that the main effect of the drug, I which was leishmanistatic, was on the duration of the 0 100 lag phase of L. mexicana cultures, whereas the effect Time in culture lhr) on T. cruzi, for which lonidamine was trypanocidal, was essentially on final cell yield. Fig. 3. Leishmanistatic effect of lonidamine. L. mexicana When the effect of lonidamine was tested on respipromastigotes were grown in the absence of lonidamine (0) ration of L. mexicana promastigotes, the inhibition or in the presence of 0.5 mM drug (A). At the time indicated was similar both when cell respiration was uncoupled by the arrow, the contents of 2 culture flasks were separately by FCCP, or when respiration was normally coupled centrifuged under sterile conditions, and the cells reto phosphorylation. This finding is again at variance suspended in fresh medium (A). Cells from another flask, resuspended in fresh medium containing 0.5 mM lowith the case of T. cruzi, where the uncoupled nidamine, did not grow (not shown). The experiment was respiration was inhibited 50% by 50 p M lonidamine, performed in duplicate, as in Fig. 2. whereas a similar inhibition of oxygen consumption in the absence of FCCP required 0.45 mM lonidamine; the latter concentration is similar to that Figure 5 presents the inhibition of respiration as a effective on L. mexicana respiration (Fig. 5). As is the function of the concentration of lonidamine. The tSO case of T. cruzi, however, there was an apparent correlation between the lonidamine concentration values were approximately 0.4 mM in the presence of effective for 50% inhibition of growth (0.42 mM) and FCCP, and 0.5mM in its absence. respiration (0.4-0.5 mM), suggesting that both are linked. The difference in effectiveness of lonidamine on both trypanosomatids is remarkable, since they DISCUSSION seem to have similar energy metabolism. Both paraLonidamine inhibited growth of L. mexicana in sites are able to ferment glucose aerobically, with culture, although the drug was less effective than in production of succinate (Hart and Coombs, 1982; the case of T. cruzi; the 150 value for L. mexicana Cazzulo et al., 1985) and their respiration is sensitive (about 0.42 mM) was twice as high as that for T. cruzi to cyanide, azide and antimycin A and insensitive to SHAM (Gutteridge and Rogerson, 1979; Hart et al., 1981). It is possible that the lower effectiveness of lonidamine on L. mexicana might be due to a lower 02 Consumptton permeability to the drug, although we can not discard an intrinsic ability of L. mexicana to detoxify
I
lonidamine; this would explain the fact that the main effect of the drug was on the lag phase of the culture, instead of on the final cell yield.
I
0
I
0.2
0.4
Lonidamine
Fig. 4. Effect of lonidamine on cell respiration before (a) and after (b) addition of the uncoupler FCCP. The numbers on the traces are rates of 0, consumption in nmol O/min (mg protein). Protein concentration was 0.4 mg/ml,
0.6
0.6
I
1.0
ImMI
Fig. 5. Inhibition of coupled (0) and FCCP-uncoupled (0) respiration of intact L. mexicana promastigotes by lonidamine. Protein concentration: 0.4 mg/ml. When present, -the concentration of FCCP was 1 FM.
JULIOF. TURRENSand JUAN Jose CAZZULO
196
Although the high lonidamine concentrations required for leishmanistatic effect make unlikely the use of this drug in the treatment of leishmaniasis, the development of more effective drug derivatives might be feasible. Acknowledgements-The authors want to thank Dr Berta Franke de Cazzulo for her assistance in some experiments. Lonidamine was kindly provided by Angelini Farmaceutici (Rome, Italy). The authors are Career Investigators from the Consejo National de Investigaciones Cientificas y Tbcnicas de la Republica Argentina.
REFERENCES Andrade F. G., Magalhaes J. B. and Pontes A. L. (1985) Evaluation of chemotherapy with benznidazol and nifurtimox in mice infected with Trypanosoma cruzi strains of different tvnes. Bull. WHO 63. 721-726. Avila A. and Muiioz E. (1981) Elects of allopurinol on different strains of Trypanosoma cruzi. Am. J. trop. med. Hyg. 30, 7699774.
Arch. Biochem. Biophys. 226, 73-83.
Floridi A., Paggi M. G., Marcante M. L., Silvestrini B., Canuto A. and De Martini C. (1981) Lonidamine, a selective inhibitor of aerobic glycolysis of murine tumor cells. J. Natl. Cancer Inst. 66, 497499. Gutteridge W. E. and Rogerson G. W. (1979) Biochemical aspects of the biology of Trypanosoma cruzi. In Biology of Kinetoplastida (Edited by Lumsden W. H. R. and Evans E. D.), Vol. 2, pp. 619-652. Academic Press, London. Hart B. T., Vickerman K. and Coombs G. H. (1981) Respiration of Leishmania mexicana amastigotes and promastigotes. Molec. Biochem. Parasitol. 4, 39-5 1. Hart B. T. and Coombs G. H. (1982) Leishmania mexicana: energy metabolism of amastigotes and promastigotes. Exp. Parasitol.
54, 397406.
Silvestrini B., Burberi S., Catanese B., Cioli V., Coulston F., Lisciani R. and Scorza Barcellona P. (1975) Antispermatogenic activity of l-a-chlorobenzyl-l,H-indazol3-carboxylic acid (AF 1312/TS) in rats. Exp. Mol. Pathol. 23, 288-307.
Band P. R., Deschamps M., Besner J. G., Laclaire R., Gervais P. and De Sanctis A. (1984) Phase I toxicologic studv, of lonidamine in cancer natients. Oncology -. 41, (suppl. 1), 5659. Cazzulo J. J., Franke de Cazzulo B. M., Engel J. C. and Cannata J. J. B. (1985) End products and enzyme levels of aerobic glucose fermentation in Trypanosomatids. Molec. Biochem. Parasitol.
Floridi A. and Lehninger A. L. (1983) Action of the antitumor and antispermatogenic agent lonidamine on electron transport in Ehrlich ascites tumor mitochondria.
16, 329-343.
Coulston F., Dougherty W. J., Le Febre R., Abraham R. and Silvestrini B. (1975) Reversible inhibition of spermatogenesis in rats and monkeys with a new class of indazol carboxylic acids. Exp. Mol. Pathol. 23, 357-366.
Silvestrini B., Palazzo G. and De Gregorio M. (1984) Lonidamine and related compounds.- In Progress in Medicinal Chemistrv (Edited bv Ellis G. P. and West G. B.), pp. 11l-135. Elsevier, North Holland, Amsterdam. Turrens J. F. (1986) Inhibitory action of the antitumor agent lonidamine on mitochondrial respiration of Trypanosoma cruzi and T. brucei. Molec. Biochem. Parasitol. 20, 237-24 1.
Young C. W., Currie V. E., Kim J. H., O’Hehir M. A., Farag F. M. and Kinahan J. E. (I 984) Phase I and clinical pharmacologic evaluation of lonidamine in patients with advanced cancer. Oncology 41 (suppl. 1) 6(t65.
0306~4492/87 $3.00+ 0.00 0 1987 Pergamon Journals Ltd
Vol. 88C, No. I, pp, 193-196, 1987
Printed in Great Britain
INHIBITION OF GROWTH AND RESPIRATION OF LEI,!$HMANIA MEXICANA BY THE ANTITUMOR AGENT LONIDAMINE JULIO F. TURRENS* and JUAN Jo& CAZZULO~ *Institute de Quimica y Fisicoqu~mica Biol6gicas, Facultad de Farmacia y Bioqu~mi~, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina; ?Instituto de Investigaciones Bioquimicas Fundaci6n Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina. Telephone: 88-1916 (Received 27 November 1986) Abstract-1. The antitumor drug lonidamine inhibited growth of promastigotes of Leishmunia mexicana in axenic culture. 2. Fifty percent inhibition was attained at 0.42 mM, and was reflected mainly in an increase in lag time, with less effect on final cell yield. 3. The drug was leishmanistatic, since when a non-growing culture in the presence of 0.5 mM lonidamine was centrifuged and the cells resuspended in fresh medium, growth started and reached the control value. 4. Both coupled and FCCP-uncoupled respiration of intact promastigotes were inhibited by lonidamine; 50% inhibition was attained at 0.5 and 0.4 mM, respectively. 5. The results suggested that the mechanism of inhibition of growth of L. mexicona is, as proposed in the case of Try~~nosvrn~cruzi epimastigotes and ~ryp~nosom~ brucei procyclic try~mastigotes, through inhibition of the energy metabolism.
INTRODUCTION Lonidamine (I-(2,4-dichlorobenzyl-1 ,H-indazol-3carboxylic acid) (Fig. 1) is a drug which was first
developed as an antis~~atogenic agent (Silvestrini et al., 1975; Coulston et al., 1975), and was later found effective against experimental tumors (Floridi et al., 1981; Silvestrini et al., 1984); it has relatively low toxicity, and clinical trials as an antitumor agent are under way (Band et al., 1984; Young et al., 1984). The mode of action of lonidamine on mammalian cells involves inhibition of energy metabolism at different levels, including the particle-bound hexokinase and uncoupled respiration (Floridi et al., 1981; Floridi and Lehninger, 1983). Recently, one of us has reported inhibition by lonidamine of growth and uncoupled respiration of Trypanosoma cruzi, the parasitic flagellate which causes the American trypanosomiasis, Chagas’ disease; uncoupled respiration of Trypanosoma brucei procyclic trypomastigotes was also inhibited by the drug, whereas the cyanidesalicyl hydroxamic acid (SHAM)insensitive, sensitive respiration of blood-stream trypomastigotes of T. brucei was much less affected (Turrens, 1986). In the present report we extend this study to another pathogenic trypanosomatid, Leishmu~ia mexicana. Growth in axenic culture of promastigotes and uncoupled respiration of the intact cells were inhibited by lonidamine, although the concentrations required were considerably higher than in the case of T. cruzi and T. brucei. Interestingly, the drug at 0.5 mM was only leishmanistatic, whereas the same concentration was clearly trypanocide against T.
University of Costa Rica, San Jo&, Costa Rica, and lr. cruzi, Tul 0 stock, were kindly given by Dr Elsa L. Segura, Instituto Fatala Chaben, Buenos Aires, Argentina. L. mexicana promastigotes and T. brucei epimastigotes were grown in a brain heart tryptose medium, containing 20% (L. mexicana) or 10% (T. cruzi) fetal calf serum (Cazzulo et al., 1985). Freshly inoculated culture medium was divided into IO-ml samples which were incubated at 25°C (L. mexicana) or 28°C (T. cruzi) in 125-ml Erlenmeyer flasks fitted with a lateral tube which allowed growth to be followed in a Lumetron Colorimeter (Photovolt Company) using a red filter. Since theese organisms form rosettes only in the stationary phase, turbidity was proportional to cell numbers. Cell motility and integrity was checked by microscopic observation. Lonidamine was added as a 50 mM solution in dimethyl-sulfoxide (DMSO). Appropriate controls indicated that even the highest DMSO concentration used (0. I in IOml of culture medium) caused negligible effects on growth per se. Respiration experiments For these experiments, promastigotes of L. mexicana were grown in the same culture medium in Roux flasks at 25”C, and harvested in the late exponential phase of growth (about 40 x 106cells/ml). The cells were harvested by centrifugation at 5800g for IS min at 4”C, and washed twice in
cruzi.
: OH clJT-+ ’ ,,i I
xl‘1
Cl
MATERIALS AND METHODS
Cl
Lonidamine
Organism and growth L. mexicana, originally isolated by Dr Rodrigo Zeledbn, C.B P. 88,1C--M
0
Fig. 1. Structure of lonidamine. 193
JULIOF. TURRENSand JUAN Jo& CAZZULO
194
shape of the inhibition curve for L. mexicana is bizarre, since 0.4mM lonidamine inhibited by 36% and 0.5mM inhibited by 100%. This was not an artifact caused by an adverse effect of the solvent, and probably reffects an extreme lengthening of the lag phase. In fact, the cells were still alive after 5 days incubation in the presence of 0.5 mM lonidamine, since when the culture medium containing the drug was removed by centrifugation and the cells resuspended in fresh medium, the promastigotes started growing after a lag phase of nearly 3 days and reached the same absorbance value as the control (Fig. 3). Microscopic observation showed that the cells had normal morphology and motility, either in the presence or in the absence of lonidamine. When an experiment similar to that of Fig. 3 was performed with T. cruzi, the cells incubated with 0.5 mM lonidamine, centrifuged and resuspended in fresh medium, did not resume growth, showing that the drug is trypanocide at this concentration (not shown). Figure 4 shows that lonidamine has an inhibitory effect on the respiration of intact L. mexicana cells, in the absence or in the presence of the uncoupler carbonyl - cyanide -p - trifluoromethoxyphenylhydra zone (FCCP).
the centrifuge with 250 mM sucrose-5 mM KCl. Oxygen consumption by the cells, suspended in the same medium, was monitored with a Clark electrode at 3o”C, using a medium containing 6mM glucose, 140mM NaCl and 10 mM Tris-HCI @H 7.4).
RESULTS Figure 2a, shows that lonidamine inhibited growth of L. mexicana in culture, although it was less effective than in the case of T. cruzi (Fig. 2b). There was a clear difference in the effects, since in the case of T. cruzi the cultures in the presence of the drug grew at a lower rate and reached a lower stationary phase, whereas in the case of L. mexicana the main effect was on the lag phase, which was progressively lengthened by increasing concentrations of the drug, with less effect on the cell concentration at the stationary phase. Figure 2c, shows cell growth as a function of lonidamine concentration; the data were taken from Fig. 2a and b. The concentration required for a 50% decrease in cell concentration of L. mexicana at the stationary phase (I~,,) was about 0.42 mM, whereas the value for T. cruzi in the present experimental
conditions
was
about
0.2 mM.
The r-
I
0
0.125 0.25 0.40
I
I
0
tb I
(dl
I
L
i” 0.2!5
0.54I_
>
I
L
I
100
20 0 Time
in
culture
Lonidamine
(hrl
ImMl
Fig. 2. Inhibition of growth of L. mexicana promastigotes (a) and T. cruzi epimastigotes Tul 0 stock (b) by lonidamine. Numbers on the curves indicate lonidamine concentration (mM). (c) Inhibition of growth of L. mexicana (0) and Z’. cruzi (0) ccl1 concentration at the stationary phase, as a function of drug concentration. The experimental points are the average of duplicates which differed at most by 10%.
Lonidamine inhibits Leishmunia
195
(0.2 mM). The latter value, obtained with the Tul 0 stock of T. cruzi, was 2.5-fold greater than that previously found with the Tul 2 stock of the same organism (Turrens, 1986). This difference might be due to strain differences, since marked differences in the effectiveness of the trypanocidal drugs nifurtimox, benznidazole and allopurinol, have been reported, depending on the parasite strain used (Andrade et al., 1985; Avila and Mufioz, 1981). Moreover, different batches of lonidamine were used in both sets of experiments. Differences in the effect of lonidamine on L. mexicana and T. cruzi are emphasized by the fact that the main effect of the drug, I which was leishmanistatic, was on the duration of the 0 100 lag phase of L. mexicana cultures, whereas the effect Time in culture lhr) on T. cruzi, for which lonidamine was trypanocidal, was essentially on final cell yield. Fig. 3. Leishmanistatic effect of lonidamine. L. mexicana When the effect of lonidamine was tested on respipromastigotes were grown in the absence of lonidamine (0) ration of L. mexicana promastigotes, the inhibition or in the presence of 0.5 mM drug (A). At the time indicated was similar both when cell respiration was uncoupled by the arrow, the contents of 2 culture flasks were separately by FCCP, or when respiration was normally coupled centrifuged under sterile conditions, and the cells reto phosphorylation. This finding is again at variance suspended in fresh medium (A). Cells from another flask, resuspended in fresh medium containing 0.5 mM lowith the case of T. cruzi, where the uncoupled nidamine, did not grow (not shown). The experiment was respiration was inhibited 50% by 50 p M lonidamine, performed in duplicate, as in Fig. 2. whereas a similar inhibition of oxygen consumption in the absence of FCCP required 0.45 mM lonidamine; the latter concentration is similar to that Figure 5 presents the inhibition of respiration as a effective on L. mexicana respiration (Fig. 5). As is the function of the concentration of lonidamine. The tSO case of T. cruzi, however, there was an apparent correlation between the lonidamine concentration values were approximately 0.4 mM in the presence of effective for 50% inhibition of growth (0.42 mM) and FCCP, and 0.5mM in its absence. respiration (0.4-0.5 mM), suggesting that both are linked. The difference in effectiveness of lonidamine on both trypanosomatids is remarkable, since they DISCUSSION seem to have similar energy metabolism. Both paraLonidamine inhibited growth of L. mexicana in sites are able to ferment glucose aerobically, with culture, although the drug was less effective than in production of succinate (Hart and Coombs, 1982; the case of T. cruzi; the 150 value for L. mexicana Cazzulo et al., 1985) and their respiration is sensitive (about 0.42 mM) was twice as high as that for T. cruzi to cyanide, azide and antimycin A and insensitive to SHAM (Gutteridge and Rogerson, 1979; Hart et al., 1981). It is possible that the lower effectiveness of lonidamine on L. mexicana might be due to a lower 02 Consumptton permeability to the drug, although we can not discard an intrinsic ability of L. mexicana to detoxify
I
lonidamine; this would explain the fact that the main effect of the drug was on the lag phase of the culture, instead of on the final cell yield.
I
0
I
0.2
0.4
Lonidamine
Fig. 4. Effect of lonidamine on cell respiration before (a) and after (b) addition of the uncoupler FCCP. The numbers on the traces are rates of 0, consumption in nmol O/min (mg protein). Protein concentration was 0.4 mg/ml,
0.6
0.6
I
1.0
ImMI
Fig. 5. Inhibition of coupled (0) and FCCP-uncoupled (0) respiration of intact L. mexicana promastigotes by lonidamine. Protein concentration: 0.4 mg/ml. When present, -the concentration of FCCP was 1 FM.
JULIOF. TURRENSand JUAN Jose CAZZULO
196
Although the high lonidamine concentrations required for leishmanistatic effect make unlikely the use of this drug in the treatment of leishmaniasis, the development of more effective drug derivatives might be feasible. Acknowledgements-The authors want to thank Dr Berta Franke de Cazzulo for her assistance in some experiments. Lonidamine was kindly provided by Angelini Farmaceutici (Rome, Italy). The authors are Career Investigators from the Consejo National de Investigaciones Cientificas y Tbcnicas de la Republica Argentina.
REFERENCES Andrade F. G., Magalhaes J. B. and Pontes A. L. (1985) Evaluation of chemotherapy with benznidazol and nifurtimox in mice infected with Trypanosoma cruzi strains of different tvnes. Bull. WHO 63. 721-726. Avila A. and Muiioz E. (1981) Elects of allopurinol on different strains of Trypanosoma cruzi. Am. J. trop. med. Hyg. 30, 7699774.
Arch. Biochem. Biophys. 226, 73-83.
Floridi A., Paggi M. G., Marcante M. L., Silvestrini B., Canuto A. and De Martini C. (1981) Lonidamine, a selective inhibitor of aerobic glycolysis of murine tumor cells. J. Natl. Cancer Inst. 66, 497499. Gutteridge W. E. and Rogerson G. W. (1979) Biochemical aspects of the biology of Trypanosoma cruzi. In Biology of Kinetoplastida (Edited by Lumsden W. H. R. and Evans E. D.), Vol. 2, pp. 619-652. Academic Press, London. Hart B. T., Vickerman K. and Coombs G. H. (1981) Respiration of Leishmania mexicana amastigotes and promastigotes. Molec. Biochem. Parasitol. 4, 39-5 1. Hart B. T. and Coombs G. H. (1982) Leishmania mexicana: energy metabolism of amastigotes and promastigotes. Exp. Parasitol.
54, 397406.
Silvestrini B., Burberi S., Catanese B., Cioli V., Coulston F., Lisciani R. and Scorza Barcellona P. (1975) Antispermatogenic activity of l-a-chlorobenzyl-l,H-indazol3-carboxylic acid (AF 1312/TS) in rats. Exp. Mol. Pathol. 23, 288-307.
Band P. R., Deschamps M., Besner J. G., Laclaire R., Gervais P. and De Sanctis A. (1984) Phase I toxicologic studv, of lonidamine in cancer natients. Oncology -. 41, (suppl. 1), 5659. Cazzulo J. J., Franke de Cazzulo B. M., Engel J. C. and Cannata J. J. B. (1985) End products and enzyme levels of aerobic glucose fermentation in Trypanosomatids. Molec. Biochem. Parasitol.
Floridi A. and Lehninger A. L. (1983) Action of the antitumor and antispermatogenic agent lonidamine on electron transport in Ehrlich ascites tumor mitochondria.
16, 329-343.
Coulston F., Dougherty W. J., Le Febre R., Abraham R. and Silvestrini B. (1975) Reversible inhibition of spermatogenesis in rats and monkeys with a new class of indazol carboxylic acids. Exp. Mol. Pathol. 23, 357-366.
Silvestrini B., Palazzo G. and De Gregorio M. (1984) Lonidamine and related compounds.- In Progress in Medicinal Chemistrv (Edited bv Ellis G. P. and West G. B.), pp. 11l-135. Elsevier, North Holland, Amsterdam. Turrens J. F. (1986) Inhibitory action of the antitumor agent lonidamine on mitochondrial respiration of Trypanosoma cruzi and T. brucei. Molec. Biochem. Parasitol. 20, 237-24 1.
Young C. W., Currie V. E., Kim J. H., O’Hehir M. A., Farag F. M. and Kinahan J. E. (I 984) Phase I and clinical pharmacologic evaluation of lonidamine in patients with advanced cancer. Oncology 41 (suppl. 1) 6(t65.