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Effect of a juvenile hormone analogue on Leishmania amazonensis and Leishmania braziliensis
Experimental Parasitology 110 (2005) 162–164 www.elsevier.com/locate/yexpr
Research brief
Effect of a juvenile hormone analogue on Leishmania amazonensis and Leishmania braziliensis M. Esteva *, C. Maidana, A. Sinagra, C. Luna, A.M. Ruiz, A.M. Stoka Instituto Nacional de Parasitologı´a ‘‘Dr. Mario Fatala Chabe´n,’’ Buenos Aires, Argentina Received 25 June 2004; received in revised form 28 January 2005; accepted 1 February 2005 Available online 19 March 2005
Leishmaniasis is a group of diseases that is present in several clinical forms as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases are caused by protozoan parasites (Leishmania spp.) which are transmitted by different species of dipteran vectors (e.g., Phlebotominae: sandflies). Improvement in treatment for human-leishmaniasis is highly desirable because the risk treatment failure through drug resistance is very high in some areas of the world such as Bihar, India, and the treatments recommended by WHO (1990) may have severe treatment limiting side-effects in patients. In consequence it is necessary to search for new antileishmanial compounds that will be pharmacologically more effective and less toxic than those already available. In this respect, there is evidence that some hormones may have originally served as defensive substances to eliminate endo- and exo-parasites in multicellular organisms (Loose et al., 1983; Schidknecht et al., 1967; Stoka et al., 1987). Furthermore, numerous observations have demonstrated that human hormones can mediate sex-associated responses to leishmaniasis infection. For instance, some experimental and epidemiological studies have shown more severe disease in females than in males (Alexander, 1988; Giannini, 1986; Greenblatt, 1980; Roberts et al., 1996; Travi et al., 2002). In concordance with these findings, it has been observed that juvenile hormone (JH), a modulator of molting, and reproductive processes in insects (Stoka, 1987), causes growth-inhibition on protozoan parasites (Clark et al., 1964; Ilan et al., 1974; Stoka, 1996; Wink, *
Corresponding author. E-mail address: [email protected] (M. Esteva).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.02.005
1979). This action, a host-hormone acting on parasites, is known as a heterotropic effect or as an exaptation of its original hormonal function (Stoka, 1999). In our laboratory, it was further observed that juvenile hormone analogues (JHA) were more effective than JH in disrupting Trypanosoma cruzi development. For instance JHA were shown to cause growth-inhibition of epimastigote forms, and also lytic effects on trypomastigote forms (Esteva et al., 2002; Fichera et al., 1995; Rodriguez et al., 1991; Schvartzapel et al., 1995; Stoka et al., 1990). It is important to note that, from a phylogenetic point of view, Trypanosoma spp. and Leishmania spp. are grouped together coming from a common origin. This is demonstrated when antigens used for diagnosis of these diseases showed significant cross-reactivity. This was observed during trials on geographical distribution of Chagas disease and leishmaniasis in some areas of Central and South America where these diseases overlap (Frank et al., 2003). The aim of this work was therefore to study ‘‘in vitro’’ the effects of the JHA methoprene (Metho), on the species of Leishmania found in the north west of Argentina, Leishmania braziliensis, and Leishmania amazonensis. Cultures used in this work were made with L. braziliensis MHOM/BR/75/ M2903, and L. amazonensis IFLA/ BR/67/PH8 (WHO, 1990) promastigotes that were grown in biphasic medium: Nutrient agar (DIFCO), 15% defibrinated blood rabbit + RPMI 1640 (Gibco-BRL Life Technologies, CA), and 20% FBS. Methoprene (isopropyl(E,E)-(RS)-11-methoxy-3,7,11-trimethyl-2,4-dienoate) was provided by Dr. Zerba (CIPEIN, Pcia. Buenos Aires, Argentina) (Fig. 1). The most pronounced inhibitory effect of Metho in L. amazonensis and L. braziliensis was observed at
M. Esteva et al. / Experimental Parasitology 110 (2005) 162–164
Fig. 1. Structure of methoprene (isopropyl(E, E)-(RS)-11 methoxy3,7,11-trimethyl-2,4-dienoate).
Table 1 Effect of methoprene on L. amazonensis and L. braziliensis promastigotes % Inhibition (X ± SD) 24 h
48 h
72 h
L. amazonensis 100 lM 43.75 ± 9.20 250 lM 74.80 ± 3.40 500 lM 83.40 ± 2.40
62.25 ± 5.97 84.50 ± 7.80 89.70 ± 7.50
51.25 ± 18.69 92.20 ± 6.10 96.60 ± 2.20
L. braziliensis 100 lM 53.00 ± 15.78 250 lM 86.60 ± 4.70 500 lM 91.10 ± 5.70
50.80 ± 4.88 93.20 ± 2.40 95.90 ± 2.90
63.00 ± 21.64 92.20 ± 4.90 97.10 ± 1.70
L. braziliensis and L. amazonensis promastigotes were incubated in BHT medium with different amounts of methoprene. The incubation was performed in 15 ml polypropylene tubes at 26 C during 72 h. In all experiments 5 ml of medium containing 2 · 105 promastigotes per milliliter and 20 ll ethanol with different quantities of methoprene were used; only ethanol was added into control samples. The concentration of ethanol (0.4%) used as control did not affect the parasites. After incubation the number of alive parasites at 24, 48, and 72 h of each sample was counted in a Neubauer chamber. Each experiment was replicated four times.
72 h (92.2 and 96%) and at 48 h (93.2 and 95.9%) with 250 and 500 lM of Metho, respectively (Table 1). In previous work, a strong lytic effect of Metho on T. cruzi trypomastigotes (in vitro) was observed at concentrations of 100 lM (30 lg/ml). A delay in detection of the parasitemia, and its decreasing and partial survival in ‘‘in vivo’’ were also observed (Esteva et al., 2002). Our studies have demonstrated that Metho is an efficient anti-leishmanial drug, although less active than in T. cruzi. In connection with the molecular mechanism of action of JH and JHA, it is known that these compounds have an extraordinarily broad range of developmental and physiological effects in different animal species (Wheeler and Nijhout, 2003). Indeed JH and JHA are members of a diverse, and wide spread class of lipid signaling molecules which can participate in signal transduction and transcriptional regulation of different regulatory processes (Wheeler and Nijhout, 2003). For instance, an exaptation of a JH-action is the inhibition of progesterone synthesis in mammalian cells, indicating that the mammalian steroidogenic pathway is affected (Vladusic et al., 1994). In correlation with the inhibition of steroidogenic pathway caused by JH, alterations of the cell
163
membrane in T. cruzi epimastigotes when exposed to high concentrations (e.g., 1 · 10 3 M) of JH or JHA (Stoka, 1996) were observed. Probably, the effects of JHA and JH in cell membranes may be correlated with the inhibition of cholesterol-biosynthesis, a key-step in the steroidogenic pathway. The low toxicity and secondary effects of Metho (oral acute LD50 for rats: 34,000 mg/kg) (FAO, 1987) suggest that Metho or other JHAs might be developed as tools for the control of leishmaniasis. Thus, the studies of the mechanism of JH and JHA action in different species might provide the basic knowledge for developing new effective and specific anti-leishmanial compounds for use in endemic areas.
References Alexander, J., 1988. Sex differences and cross-immunity in DBA/2 mice infected with L. mexicana and L. major. Parasitology 96, 297– 302. Clark, T.B., Kellen, W.R., Lindegren, J.E., 1964. The transmission of Crithidia fasciculata (Leger, 1902) in Culiseta incidens. Journal of Protozoology 11, 400–402. Esteva, M., Ruiz, A.M., Stoka, A.M., 2002. Trypanosoma cruzi: methoprene is a potent agent to sterilize blood infected with trypomastigotes. Experimental Parasitology 100, 248–251. FAO, 1987. Pesticide residues in food. FAO Plan Production and Protection. Paper 84. Fichera, L., Esteva, M., Wimmer, Z., Rodrı´guez, J.B., Gros, E.G., 1995. Effects of juvenile hormone analogues (JHA) on the development of Trypanosoma cruzi. Zeitschrift fur Naturforschung 50c, 578–580. Frank, F.M., Fernandez, M., Taranto, N.J., Cajal, S.P., Margni, R.A., Castro, E., Thomaz-Soccol, Malchiodi, E.L., 2003. Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved. Parasitology 126, 31–39. Giannini, M.S., 1986. Sex-influenced response in the pathogenesis of cutaneous leishmaniasis in mice. Parasite Immunology 8, 31–37. Greenblatt, C.L., 1980. The present and future of vaccination for cutaneous leishmanaisis. Progress in Clinical and Biological Research 47, 259–285. Ilan, J., Ilan, J., Rickles, S., 1974. Inhibition by juvenile hormone of growth of Crithidia fasciculata in culture. Nature 224, 179–180. Loose, D.S., PriceStover, E., Restrepo, A., Stevens, D.A., Feldman, D., 1983. Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis. Proceedings of the National Academy of Sciences of the United States of America 80, 7659–7663. Roberts, C.W., Satoskar, A., Alexander, J., 1996. Sex steroids pregnancy-associated hormones and immunity to parasitic infection. Parasitology Today 12, 382–388. Rodriguez, J.B., Gros, E.G., Stoka, A.M., 1991. Synthesis and activity of juvenile hormone analogues (JHA) for Trypasoma cruzi. Bioorganic and Medicinal Chemistry Letters 1, 679–682. Schidknecht, H., Birringer, H., Maschwitz, U., 1967. Testosterone as protective agent of the water beetle Ilybius. Angewandte Chemie International Edition (English) 6, 558. Schvartzapel, A.J., Fichera, L., Esteva, M., Rodrı´guez, J.B., Gros, E.G., 1995. Design, sintesis and anti-Trypanosoma cruzi evaluation of a new class of cell-growth inhibitors structurally related to fenoxycarb. Helvetica Chimica Acta 78, 1207–1214.
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M. Esteva et al. / Experimental Parasitology 110 (2005) 162–164
Stoka, A.M., 1987. Ecdysteroids, juvenile hormones and metamorphosis. In: Stoka, A.M., Brenner, R.R. (Eds.), Chagas Disease Vectors, vol. III. CRC Press, Florida, USA, pp. 71–99. Stoka, A.M., Ronderos, J.R., Reboredo, G.R., 1987. Insect control by endocrine disorder. In: Brenner, R.R., Stoka, A.M. (Eds.), Chagas Disease Vectors, vol. III. CRC Press, Florida, USA, pp. 139–147. Stoka, A.M., Rivas, C., Segura, E.L., Rodrı´guez, J.B., Gros, E.G., 1990. Biological activity of synthetic juvenile hormone analogues (JHA) for Trypanosoma cruzi. Zeitschrift fur Naturforschung 45b, 96–98. Stoka, A.M., 1996. Activity of juvenile hormone and juvenile hormone analogues on the growth of Trypanosoma cruzi. Journal of Steroid Biochemistry and Molecular Biology 59, 495–500. Stoka, A.M., 1999. Phylogeny and evolution of chemical communication: an endocrine approach. Journal of Molecular Endocrinology 22, 207–225.
Travi, B.L., Osoria, Y., Melby, P.C., Chandrasekar, B., Arteaga, L., Sarabia, N.G., 2002. Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp. Infection and Immunity 70, 2288–2296. Vladusic, E.A., Bussmann, L.E., Visconti, P.E., Stoka, A.M., Rodriguez, J.B., Gros, E.G., Charreau, E.H., 1994. Effects of juvenile hormone on mammalian steroidogenesis. Journal of Steroid Biochemistry and Molecular Biology 50, 181–187. Wheeler, D.E., Nijhout, H.F., 2003. A perspective for understanding the models of JH action as a lipid signaling system. BioEssays 25, 994–1001. WHO, 1990 Control of the Leishmaniasis, 1990. Report of a WHO Expert Committee. Technical Report Series, N0 793. WHO, Geneva. Wink, M., 1979. Trypanosoma theileri: in vitro cultivation in tse-tse fly and vertebrate cell culture systems. International Journal of Parasitology 9, 585–589.
Research brief
Effect of a juvenile hormone analogue on Leishmania amazonensis and Leishmania braziliensis M. Esteva *, C. Maidana, A. Sinagra, C. Luna, A.M. Ruiz, A.M. Stoka Instituto Nacional de Parasitologı´a ‘‘Dr. Mario Fatala Chabe´n,’’ Buenos Aires, Argentina Received 25 June 2004; received in revised form 28 January 2005; accepted 1 February 2005 Available online 19 March 2005
Leishmaniasis is a group of diseases that is present in several clinical forms as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases are caused by protozoan parasites (Leishmania spp.) which are transmitted by different species of dipteran vectors (e.g., Phlebotominae: sandflies). Improvement in treatment for human-leishmaniasis is highly desirable because the risk treatment failure through drug resistance is very high in some areas of the world such as Bihar, India, and the treatments recommended by WHO (1990) may have severe treatment limiting side-effects in patients. In consequence it is necessary to search for new antileishmanial compounds that will be pharmacologically more effective and less toxic than those already available. In this respect, there is evidence that some hormones may have originally served as defensive substances to eliminate endo- and exo-parasites in multicellular organisms (Loose et al., 1983; Schidknecht et al., 1967; Stoka et al., 1987). Furthermore, numerous observations have demonstrated that human hormones can mediate sex-associated responses to leishmaniasis infection. For instance, some experimental and epidemiological studies have shown more severe disease in females than in males (Alexander, 1988; Giannini, 1986; Greenblatt, 1980; Roberts et al., 1996; Travi et al., 2002). In concordance with these findings, it has been observed that juvenile hormone (JH), a modulator of molting, and reproductive processes in insects (Stoka, 1987), causes growth-inhibition on protozoan parasites (Clark et al., 1964; Ilan et al., 1974; Stoka, 1996; Wink, *
Corresponding author. E-mail address: [email protected] (M. Esteva).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.02.005
1979). This action, a host-hormone acting on parasites, is known as a heterotropic effect or as an exaptation of its original hormonal function (Stoka, 1999). In our laboratory, it was further observed that juvenile hormone analogues (JHA) were more effective than JH in disrupting Trypanosoma cruzi development. For instance JHA were shown to cause growth-inhibition of epimastigote forms, and also lytic effects on trypomastigote forms (Esteva et al., 2002; Fichera et al., 1995; Rodriguez et al., 1991; Schvartzapel et al., 1995; Stoka et al., 1990). It is important to note that, from a phylogenetic point of view, Trypanosoma spp. and Leishmania spp. are grouped together coming from a common origin. This is demonstrated when antigens used for diagnosis of these diseases showed significant cross-reactivity. This was observed during trials on geographical distribution of Chagas disease and leishmaniasis in some areas of Central and South America where these diseases overlap (Frank et al., 2003). The aim of this work was therefore to study ‘‘in vitro’’ the effects of the JHA methoprene (Metho), on the species of Leishmania found in the north west of Argentina, Leishmania braziliensis, and Leishmania amazonensis. Cultures used in this work were made with L. braziliensis MHOM/BR/75/ M2903, and L. amazonensis IFLA/ BR/67/PH8 (WHO, 1990) promastigotes that were grown in biphasic medium: Nutrient agar (DIFCO), 15% defibrinated blood rabbit + RPMI 1640 (Gibco-BRL Life Technologies, CA), and 20% FBS. Methoprene (isopropyl(E,E)-(RS)-11-methoxy-3,7,11-trimethyl-2,4-dienoate) was provided by Dr. Zerba (CIPEIN, Pcia. Buenos Aires, Argentina) (Fig. 1). The most pronounced inhibitory effect of Metho in L. amazonensis and L. braziliensis was observed at
M. Esteva et al. / Experimental Parasitology 110 (2005) 162–164
Fig. 1. Structure of methoprene (isopropyl(E, E)-(RS)-11 methoxy3,7,11-trimethyl-2,4-dienoate).
Table 1 Effect of methoprene on L. amazonensis and L. braziliensis promastigotes % Inhibition (X ± SD) 24 h
48 h
72 h
L. amazonensis 100 lM 43.75 ± 9.20 250 lM 74.80 ± 3.40 500 lM 83.40 ± 2.40
62.25 ± 5.97 84.50 ± 7.80 89.70 ± 7.50
51.25 ± 18.69 92.20 ± 6.10 96.60 ± 2.20
L. braziliensis 100 lM 53.00 ± 15.78 250 lM 86.60 ± 4.70 500 lM 91.10 ± 5.70
50.80 ± 4.88 93.20 ± 2.40 95.90 ± 2.90
63.00 ± 21.64 92.20 ± 4.90 97.10 ± 1.70
L. braziliensis and L. amazonensis promastigotes were incubated in BHT medium with different amounts of methoprene. The incubation was performed in 15 ml polypropylene tubes at 26 C during 72 h. In all experiments 5 ml of medium containing 2 · 105 promastigotes per milliliter and 20 ll ethanol with different quantities of methoprene were used; only ethanol was added into control samples. The concentration of ethanol (0.4%) used as control did not affect the parasites. After incubation the number of alive parasites at 24, 48, and 72 h of each sample was counted in a Neubauer chamber. Each experiment was replicated four times.
72 h (92.2 and 96%) and at 48 h (93.2 and 95.9%) with 250 and 500 lM of Metho, respectively (Table 1). In previous work, a strong lytic effect of Metho on T. cruzi trypomastigotes (in vitro) was observed at concentrations of 100 lM (30 lg/ml). A delay in detection of the parasitemia, and its decreasing and partial survival in ‘‘in vivo’’ were also observed (Esteva et al., 2002). Our studies have demonstrated that Metho is an efficient anti-leishmanial drug, although less active than in T. cruzi. In connection with the molecular mechanism of action of JH and JHA, it is known that these compounds have an extraordinarily broad range of developmental and physiological effects in different animal species (Wheeler and Nijhout, 2003). Indeed JH and JHA are members of a diverse, and wide spread class of lipid signaling molecules which can participate in signal transduction and transcriptional regulation of different regulatory processes (Wheeler and Nijhout, 2003). For instance, an exaptation of a JH-action is the inhibition of progesterone synthesis in mammalian cells, indicating that the mammalian steroidogenic pathway is affected (Vladusic et al., 1994). In correlation with the inhibition of steroidogenic pathway caused by JH, alterations of the cell
163
membrane in T. cruzi epimastigotes when exposed to high concentrations (e.g., 1 · 10 3 M) of JH or JHA (Stoka, 1996) were observed. Probably, the effects of JHA and JH in cell membranes may be correlated with the inhibition of cholesterol-biosynthesis, a key-step in the steroidogenic pathway. The low toxicity and secondary effects of Metho (oral acute LD50 for rats: 34,000 mg/kg) (FAO, 1987) suggest that Metho or other JHAs might be developed as tools for the control of leishmaniasis. Thus, the studies of the mechanism of JH and JHA action in different species might provide the basic knowledge for developing new effective and specific anti-leishmanial compounds for use in endemic areas.
References Alexander, J., 1988. Sex differences and cross-immunity in DBA/2 mice infected with L. mexicana and L. major. Parasitology 96, 297– 302. Clark, T.B., Kellen, W.R., Lindegren, J.E., 1964. The transmission of Crithidia fasciculata (Leger, 1902) in Culiseta incidens. Journal of Protozoology 11, 400–402. Esteva, M., Ruiz, A.M., Stoka, A.M., 2002. Trypanosoma cruzi: methoprene is a potent agent to sterilize blood infected with trypomastigotes. Experimental Parasitology 100, 248–251. FAO, 1987. Pesticide residues in food. FAO Plan Production and Protection. Paper 84. Fichera, L., Esteva, M., Wimmer, Z., Rodrı´guez, J.B., Gros, E.G., 1995. Effects of juvenile hormone analogues (JHA) on the development of Trypanosoma cruzi. Zeitschrift fur Naturforschung 50c, 578–580. Frank, F.M., Fernandez, M., Taranto, N.J., Cajal, S.P., Margni, R.A., Castro, E., Thomaz-Soccol, Malchiodi, E.L., 2003. Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved. Parasitology 126, 31–39. Giannini, M.S., 1986. Sex-influenced response in the pathogenesis of cutaneous leishmaniasis in mice. Parasite Immunology 8, 31–37. Greenblatt, C.L., 1980. The present and future of vaccination for cutaneous leishmanaisis. Progress in Clinical and Biological Research 47, 259–285. Ilan, J., Ilan, J., Rickles, S., 1974. Inhibition by juvenile hormone of growth of Crithidia fasciculata in culture. Nature 224, 179–180. Loose, D.S., PriceStover, E., Restrepo, A., Stevens, D.A., Feldman, D., 1983. Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis. Proceedings of the National Academy of Sciences of the United States of America 80, 7659–7663. Roberts, C.W., Satoskar, A., Alexander, J., 1996. Sex steroids pregnancy-associated hormones and immunity to parasitic infection. Parasitology Today 12, 382–388. Rodriguez, J.B., Gros, E.G., Stoka, A.M., 1991. Synthesis and activity of juvenile hormone analogues (JHA) for Trypasoma cruzi. Bioorganic and Medicinal Chemistry Letters 1, 679–682. Schidknecht, H., Birringer, H., Maschwitz, U., 1967. Testosterone as protective agent of the water beetle Ilybius. Angewandte Chemie International Edition (English) 6, 558. Schvartzapel, A.J., Fichera, L., Esteva, M., Rodrı´guez, J.B., Gros, E.G., 1995. Design, sintesis and anti-Trypanosoma cruzi evaluation of a new class of cell-growth inhibitors structurally related to fenoxycarb. Helvetica Chimica Acta 78, 1207–1214.
164
M. Esteva et al. / Experimental Parasitology 110 (2005) 162–164
Stoka, A.M., 1987. Ecdysteroids, juvenile hormones and metamorphosis. In: Stoka, A.M., Brenner, R.R. (Eds.), Chagas Disease Vectors, vol. III. CRC Press, Florida, USA, pp. 71–99. Stoka, A.M., Ronderos, J.R., Reboredo, G.R., 1987. Insect control by endocrine disorder. In: Brenner, R.R., Stoka, A.M. (Eds.), Chagas Disease Vectors, vol. III. CRC Press, Florida, USA, pp. 139–147. Stoka, A.M., Rivas, C., Segura, E.L., Rodrı´guez, J.B., Gros, E.G., 1990. Biological activity of synthetic juvenile hormone analogues (JHA) for Trypanosoma cruzi. Zeitschrift fur Naturforschung 45b, 96–98. Stoka, A.M., 1996. Activity of juvenile hormone and juvenile hormone analogues on the growth of Trypanosoma cruzi. Journal of Steroid Biochemistry and Molecular Biology 59, 495–500. Stoka, A.M., 1999. Phylogeny and evolution of chemical communication: an endocrine approach. Journal of Molecular Endocrinology 22, 207–225.
Travi, B.L., Osoria, Y., Melby, P.C., Chandrasekar, B., Arteaga, L., Sarabia, N.G., 2002. Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp. Infection and Immunity 70, 2288–2296. Vladusic, E.A., Bussmann, L.E., Visconti, P.E., Stoka, A.M., Rodriguez, J.B., Gros, E.G., Charreau, E.H., 1994. Effects of juvenile hormone on mammalian steroidogenesis. Journal of Steroid Biochemistry and Molecular Biology 50, 181–187. Wheeler, D.E., Nijhout, H.F., 2003. A perspective for understanding the models of JH action as a lipid signaling system. BioEssays 25, 994–1001. WHO, 1990 Control of the Leishmaniasis, 1990. Report of a WHO Expert Committee. Technical Report Series, N0 793. WHO, Geneva. Wink, M., 1979. Trypanosoma theileri: in vitro cultivation in tse-tse fly and vertebrate cell culture systems. International Journal of Parasitology 9, 585–589.