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Effect of usnic acid from the lichen Cladonia substellata on Trypanosoma cruzi in vitro: an ultrastructural study
Micron 36 (2005) 155–161 www.elsevier.com/locate/micron
Effect of usnic acid from the lichen Cladonia substellata on Trypanosoma cruzi in vitro: an ultrastructural study E.A.B. De Carvalhoa, P.P. Andradeb, N.H. Silvac, E.C. Pereirad, R.C.B.Q. Figueiredoe,* a
Laborato´rio de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-901 Recife, PE, Brazil b Departamento de Gene´tica, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-901 Recife, PE, Brazil c Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-420 Recife, PE, Brazil d Departamento de Cieˆncias Geograficas; Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-901 Recife, PE, Brazil e Departamento de Biologia Celular e Ultraestrutura, Centro de Pesquisas Aggeu Magalha˜es/FIOCRUZ, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-420 Recife, PE, Brazil Received 7 June 2004; revised 16 September 2004; accepted 16 September 2004
Abstract Chemotherapy for Chagas’ disease is still unsatisfactory due to toxicity and limited effectiveness of the available drugs. In this work we have investigated the effect of usnic acid, isolated from lichen Cladonia substellata, against Trypanosoma cruzi, in vitro. Incubation of culture epimastigotes with 5–30 mg/ml of this compound resulted in growth inhibition in a dosis-dependent manner. Ultrastructural analysis of treated epimastigotes showed damage to mitochondria, with a marked increase in kinetoplast volume and vacuolation of the mitochondrial matrix. Intense lysis of bloodstream trypomastigotes was observed with all drug concentrations tested. Besides mitochondrial and kinetoplast damage, trypomastigotes also presented enlargement of the flagellar pocket, as well as intense cytoplasm vacuolation. Treatment of infected macrophages with 40 or 80 mg/ml usnic acid induced marked cytoplasm vacuolation in intracellular amastigote forms, with disorganization of parasite kinetoplast and mitochondria, but with no significant ultrastructural damage to the host cells. q 2004 Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Cladonia substellata; Usnic acid; Ultrastructure
1. Introduction Chagas’ disease is a serious health problem in South America, particularly in Brazil. More than 5 million Brazilians are affected, with major looses in work ability and life quality (WHO, 1990; Moncayo, 1993). Etiological agent of this disease is the haemoflagellate protozoan Trypanosoma cruzi, which is transmitted to humans and other mammals by triatomine bugs, with further human infection occurring by blood transfusion and transplacentally. Therapy for Chagas’ disease is unsatisfactory, due to the significant toxicity of the available drugs (nifurtimox and benznidazole). Although both compounds may reduce the acute phase and decrease mortality, parasitological cure is achieved in only 60% of acute patients and their use * Corresponding author. Tel.: C55 81 21012556; fax: C55 81 34532449. E-mail address: [email protected] (R.C.B.Q. Figueiredo). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.09.003
during the chronic phase of the disease is a matter of considerable discussion. (De Castro, 1993; Estani et al., 1998). Furthermore, differences in the susceptibility of T. cruzi strains to these drugs have been reported (Filardi et al., 1987). Lichens are symbiotic associations between fungal (mycobiont) and algal or cyanobacterial (photobiont) partners. They are able to synthesize several metabolites, comprising aliphatic, cycloaliphatic, aromatic and terpenic compounds (Huneck, 1999). Lichens have been used for medicinal purposes throughout the ages: some of them, as Cetraria islandica, Lobaria pulmonaria and Cladonia species, were reported to be effective in the treatment of pulmonary tuberculosis (Vartia, 1973). Usnic acid, [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)dibenzo-furandione; C18H16O7], is a yellow cortical pigment found only in lichens, occurring under two enantiomeric forms. Besides its antimicrobial activity
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against human and plant pathogens, usnic acid exhibits antiproliferative, anti-inflammatory and analgesic activities. (Al-Bekairi et al., 1991; Okuyama et al., 1995; Lauterwein et al., 1995; Ingolfsdottir et al., 1998; Ogmundsdottir et al., 1998). Previous studies have shown that usnic acid can cause the uncoupling of the oxidative phosphorilation of mitochondria (Abo-Khatwa et al., 1996), and thereby it has been used as a fat burner. On the other hand, deleterious effects of usnic acid on mammalian cells have been reported, by inhibiting cell proliferation and causing cytotoxicity in cultured keratinocytes (Kumar and Muller, 1999). Treatment of mice with usnic acid inhibited the proliferation of polychromatic erythrocytes (Al-Bekairi et al., 1991). Despite the potential use of usnic acid and other lichen compounds as chemotherapic drugs, few investigations have been performed on their activity against parasitic protozoa. It has been shown showed that (K)usnic acid exhibited a strong effect against Trichomonas vaginalis in vitro (Wu et al., 1995). Intralesional administration of usnic acid in BALB/c mice infected with promastigote forms of Leishmania, produced a significant reduction of cutaneous lesion (Fournet et al., 1997). The aim of this study was to analyze, at the ultrastructural level, the in vitro effects of usnic acid extracted from Cladonia substellata against the protozoan T. cruzi, the etiological agent of Chagas’ disease.
was spectrophotometrically estimated at 630 nm, in duplicate. Optical density was assumed to be directly correlated to parasite number in culture medium. Epimastigotes growing in LIT medium containing 1% DMSO were used as a control. Statistical analysis of growth differences between treated and control cultures was performed using the Wilcoxon Test, with p!0.05. After 5 days, control and treated parasites were harvested by centrifugation and processed for transmission electron microscopy, as described below.
2. Materials and methods
2.5. Effect of usnic acid on amastigotes
2.1. Parasites
Peritoneal macrophages were obtained from Swiss Webster mice as described elsewhere (Araujo-Jorge et al., 1989) Macrophage cultures were maintained with RPMI medium and then infected with bloodstream trypomastigotes at a parasite/cell ratio of 10:1. After 1 h, noninternalized parasites were washed out and fresh RPMI medium was added. Three days after infection, fresh medium with different concentrations of usnic acid (20, 40 and 80 mg/ml) was added and cells were cultivated for additional 24 h. At the end of the incubation time, control and treated–infected cells were fixed and processed for electron microscopy.
T. cruzi epimastigotes (Dm28c clone) were axenically maintained at 28 8C in LIT medium supplemented with 10% fetal bovine serum (Camargo, 1964) and harvested at the exponential phase of growth. Bloodstream trypomastigotes (Y strain) were obtained from infected albino Swiss mice at the peak of parasitemia, as described elsewhere (Meirelles et al., 1984). Intracellular amastigotes were obtained by infection of mouse peritoneal macrophages as described below.
2.4. Effect of usnic acid on bloodstream trypomastigotes Bloodstream trypomastigote forms were ressuspended in RPMI medium (Sigma Chemical Co., USA) supplemented with 2.5% fetal bovine serum and inoculated in 24-well plates at 4.3!105 cells/ml, in absence or presence of 5, 10, 20,40 and 80 mg/ml usnic acid. The parasites were cultivated for 72 h at 37 8C and the number of viable trypomastigotes/ml was estimated by daily counting using a haemocytometer. Counts were performed in triplicate and statistically analyzed with the Student’s t test, with p!0.005. For ultrastructural analysis, bloodstream trypomastigotes forms were ressuspended in RPMI medium, incubated in absence or presence of 20 mg/ml of usnic acid for 24 h at 37 8C and then processed for transmission electron microscopy.
2.2. Drugs 2.6. Transmission electron microscopy Usnic acid was isolated from the crude extract of the lichen C. substellata. Fractionation and purification of this compound were performed as previously described (Pereira et al., 1995). A stock solution was prepared at 1 mg/ml in 1% DMSO and kept at K20 8C until use. 2.3. Effect of usnic acid on epimastigotes Different drug concentrations (5, 10, 20, 30, 40, 50 mg/ml, diluted in LIT medium) were added to cell culture flasks containing 2!106 cells/ml. Aliquots (1 ml) were daily collected during five days and the culture growth
Control and treated culture epimastigotes, bloodstream trypomastigotes and infected macrophages were harvested, at room temperature by centrifugation at 1500g, washed twice in PBS and fixed for 2 h at 4 8C in a solution containing 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M cacodylate buffer, pH 7.2. After washing in this same buffer, cells were post-fixed for 1 h in a solution containing 1% OsO4, 0.8% potassium ferricyanide and 5 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.2. The cells were then dehydrated in acetone and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead
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citrate and examined in a Zeiss EM 109 transmission electron microscope.
3. Results Incubation of epimastigote cultures of T. cruzi with different concentrations of usnic acid resulted in a strong decrease of O.D. values, in a dosis-dependent manner. When 5 mg/ml of usnic acid were added to the medium, slight growth inhibition was observed only after 48 h of cultivation. However, the inhibitory effect on parasite proliferation was higher and statistically significant at concentrations ranging from 10 to 50 mg/ml. (Fig. 1). Changes in cell morphology, including increase in cell volume, loss of cell polarity due to nuclear and kinetoplast misplacement, and abnormally shaped body were observed in several parasites, as seen by light microscopy (data not shown). These effects were observed with all drug concentrations tested, but the number of affected cells and the severity of morphological changes depended on the drug concentration. Incubation of control epimastigotes with DMSO alone resulted in no significant effect on cell growth and morphology. Observation of untreated epimastigotes by transmission electron microscopy showed a centrally located nucleus, a single elongated mitochondrion running along the cell body, a compact condensation of mitochondrial kinetoplast DNA and reservosomes, the latter corresponding to the prelysosomal compartments in this parasite (Fig. 2). Ultrastructural analysis of epimastigotes treated with 5–10 mg/ml
Fig. 1. Inhibitory effects of usnic acid on epimastigote growth: (&) control; (%) 5 mg/ml; (C) 10 mg/ml; (:) 20 mg/ml; (*) 30 mg/ml; (,) 40 mg/ml and (B) 50 mg/ml. The points represent the mean value of two independent measurements.
Fig. 2. Ultrastructure of control epimastigote form grown in LIT medium. Longitudinal section showing reservosomes (R) at the posterior end, the centrally located nucleus (N) and rod-shaped kinetoplast (K). Mitochondrial profiles (arrow) are observed immediately beneath the plasma membrane. Bar: 0.5 mm.
usnic acid showed small changes in the cytoplasm density, cell swelling and loss of cell polarity (Fig. 3). Marked swelling and branching of mitochondria (Fig. 4), as well as loss of organization of the mitochondrial cristae, could be observed in cells treated with 30–50 mg/ml usnic acid. Highly condensed chromatin and severe vacuolation of mitochondria were usually seen at higher drug concentrations (Fig. 5). The Golgi apparatus, the endoplasmic
Fig. 3. Low magnification of epimastigote culture treated with 5 mg/ml of usnic acid showing eletron-luscent cytoplasm (*) and loss of cell polarity. Reservosomes (arrow) are spread throughout the cytoplasm. Bar: 5 mm.
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Fig. 4. Pronounced mitochondrial branching (m) can be observed throughout the cytoplasm of an epimastigote form incubated with 30 mg/ml usnic acid. Bar: 0.25 mm.
reticulum and the reservosomes remained essentially unaffected. Bloodstream trypomastigotes remained motile and with no visible changes in cell shape for the first 15 h in the presence of usnic acid, with all concentrations tested. However, 100% cell death was observed at 24 h for 40 and 80 mg/ml usnic acid, as well as at 48 and 72 h for drug
Fig. 5. Electron microscopy of epimastigote form treated with 50 mg/ml usnic acid showing marked vacuolation (arrow) of mitochondrial matrix and blebbing of mitochondrial membrane (arrowhead) Changes in the nucleus (N), including electron-luscent appearance of nuclear matrix and chromatin condensation can be observed. Bar: 0.25 mm.
Fig. 6. Effect of different concentrations of usnic acid on bloodstream tripomastigote viability in vitro. (C) 40 and 80 mg/ml, (!) 20 mg/ml, (:) 10 mg/ml, (&) 5 mg/ml, (%) controlCDMSO. Each point is the mean of three independent countings. All values are given as meanGSD. For all concentrations the effect of usnic acid on bloodstream trypomastigotes was statically significant (p!0.05).
concentrations of 20 and 10 mg/ml, respectively. A progressive decrease of trypomastigote viability was observed when parasites were incubated with 5 mg/ml usnic acid, reaching about 72% parasite death after 72 h of cultivation (Fig. 6). At the ultrastructural level, untreated bloodstream
Fig. 7. Effect of usnic acid treatment on bloodstream trypomastigotes of T. cruzi. Ultrastructure appearance of a control bloodstream trypomastigote (T) showing well preserved mitochondrion (arrow) and homogeneous cytoplasm.
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of the drug and changes induced by the drug treatment were investigated by both light and electron microscopy. In untreated macrophages, well preserved trypomastigotes and amastigotes could be observed in the host cell cytoplasm (Fig. 9a). No major alteration in this pattern was observed in infected cells treated with 20 mg usnic acid for 24 h. On the other hand, incubation with 40 and 80 mg/ml caused marked changes on the parasite ultrastructure. Amastigote forms exhibited mitochondrial and kinetoplast swelling and intense vacuolation (Fig. 9b), as also observed with the trypomastigote and epimastigote forms. An electron-luscent space with membrane profiles could be observed between the parasite and the host cell cytoplasm. At 80 mg/ml no intact intracellular parasites could be found (Fig. 9b), but only minor ultrastructural changes were detected in mitochondria, endoplasmic reticulum and other organelles of host cells. The number of infected cell dropped significantly when observed by light microscopy (data not shown).
Fig. 8. Effect of usnic acid treatment on bloodstream trypomastigotes of T. cruzi. Incubation with 20 mg/ml usnic acid induces an increase of mitochondrial (m) and kinetoplast (K) volume, loss of cytoplasmic content and vacuolation (*). Bar: 1 mm.
trypomastigotes presented a small flagellar pocket, an anterior flagellum, several electron-dense granules, a single mitochondrion (Fig. 7), and a characteristic basket-shaped kinetoplast. In treated trypomastigotes the major morphological changes were observed in the mitochondrion and kinetoplast (Fig. 8), although considerable swelling of the flagellar pocket and intense cytoplasmic vacuolation were also observed. Round shaped parasites were usually found. In order to analyze the ultrastructural effects of usnic acid on the clinically relevant intracellular amastigote forms, infected macrophages were treated with 20, 40 and 80 mg/ml
4. Discussion Usnic acid, which exists in nature in both (C) and (K) forms, is the most abundant constituent of several lichen species, including those belonging to the Usnea and Cladonia genera (Nishtoba et al., 1987) This compound is known to present several biological activities, acting as an anti-microbial, anti-tumoral and anti-inflammatory agent (Cocchietto et al., 2002). Our data demonstrate for the first time the effects of usnic acid on the protozoan T. cruzi. We initially tested the effect of several usnic acid concentrations on the epimastigotes proliferation in vitro. The strong decrease of O.D. values found in usnic acid treated cultures points to an effect on both growth and viability of epimastigote forms. The growth inhibition pattern was similar to that found when a crude extract of
Fig. 9. Effect of usnic acid on amastigotes. (a) Untreated macrophage showing well preserved parasites (arrow). Bar: 1 mm. (b) T. cruzi-infected macrophage treated with 80 mg/ml usnic acid for 24 h. Note the parasite disorganization, with swelling of the mitochondrion and the kinetoplast (*), as well as eletronluscent appearance of the nucleus (N) and chromatin condensation. Bar: 0.5 mm.
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C. substellata, containing about 90% usnic acid (Ahti et al., 1993; Pereira et al., 1994) was used (unpublished data). The morphological effects observed on mitochondria and kinetoplast suggest that the drug affects the energy metabolism of epimastigote. Indeed, it has been suggested that usnic acid acts as an uncoupler of oxidative phosphorylation in mouse liver mitochondria (Abo-Khatwa et al., 1996). Furthermore, the lipophylic (Muller, 2001) and ionization characteristics of this compound may alter the mitochondrial membranes, causing the organelle collapse. No significant difference in culture growth was detected at 5 mg/ml, when compared to the control. The weak effect observed at 5 mg/ml may be due to the existence of glycosomes in T. cruzi, which are organelles involved in the glycolytic pathway in trypanosomatids (Opperdoes and Michels, 1993). It is possible that at lower drug concentrations, glycosomes supply the deficiency in energy caused by the mitochondrial damage. Incubation of bloodstream trypomastigotes with usnic acid resulted in lysis of parasites, even at low drug concentrations. Although the ultrastructural observations suggest that mitochondrion and kinetoplast were the targets for usnic acid, as demonstrated for epimastigote forms, cytoplasmic vacuolation and swelling of the flagellar pocket were usually found in bloodstream trypomastigotes. These additional effects suggest that trypomastigotes are more susceptible to usnic acid treatment, probably due to differences in membrane permeability between the two forms (De Souza, 1999). Amastigotes were also affected by usnic acid treatment, although the drug concentration needed to achieve mitochondria and kinetoplast swelling were higher than those used for epimastigote and trypomastigote forms. Contrary to the high toxicity reported in the literature (Han et al., 2004; Pramyothin et al., 2004), the use of usnic acid at 80 mg/ml for 24 h was not toxic to murine peritoneal macrophages, since no significant alterations on cell morphology and ultrastructure could be observed under these conditions. Furthermore, no side effects were observed in T. cruzi-infected mice treated for 5 days with 25 mg/kg/day of usnic acid (unpublished data). Usnic acid has been shown to interfere with normal mitochondrial metabolism (Han et al., 2004), which may cause an increase in reactive oxygen species leading to both parasite and host cell death. However, the drug concentration needed to damage the host cell is considerably higher than those used in our experiments. T. cruzi is deficient in the detoxification mechanism of oxygen metabolites, and therefore is more sensitive to oxidative stress than vertebrate cells (Schirmer et al., 1987). In addition, the combination of lower drug concentrations with reduced time of treatment could help to solve this question. Furthermore, usnic acid incorporation into nanoparticles of biocompatible polymers is currently underway in our laboratory in an attempt to overcome the deleterious effect of this compound on the host cells.
In conclusion, although further studies are needed in order to elucidate the pathophysiological effects of usnic acid, either in vivo or in vitro, our results point towards the potential use of usnic acid as a chemotherapic agent for Chagas’ disease.
Acknowledgements The authors would like to thank Mr Raimundo Nazareno C. Pimentel for his technical assistance. This work was supported by CPqAM/FIOCRUZ, and UFPE.
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E.A.B. De Carvalho et al. / Micron 36 (2005) 155–161 Ingolfsdottir, K., Chung, G.A., Skulason, V.G., Gissurarson, S.R., Vilhelmsdottir, M., 1998. Antimycobacterial activity of lichen metabolites in vitro. European Journal of Pharmaceutical Science 6, 141–144. Kumar, K.C., Muller, K., 1999. Lichen metabolites, antiproliferative and cytotoxic activity of gyrophoric, usnic, and diffractaic acid on human keratinocyte growth. Journal of Natural Products 62, 821–823. Lauterwein, M., Oethinger, M., Belsner, K., Peters, T., Marre, R., 1995. In vitro activities of the lichen secondary metabolites vulpinic acid, (C)usnic acid, and (K)-usnic acid against aerobic and anaerobic microorganisms. Antimicrobial Agents and Chemotherapy 39, 2541– 2543. Meirelles, M.N., Souto-Padron, T., De Souza, W., 1984. Participation of cell surface anionic sites in the interaction between Trypanosoma cruzi and macrophages. Journal of Submicroscopy Cytology and Pathology 16, 533–545. Moncayo, A., 1993. Program Report of the UNPD/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). World Health Organization, Geneva pp. 67–75. Muller, K., 2001. Pharmaceutically relevant metabolites from lichens. Applied Microbiology and Biotechnology 56, 9–16. Nishtoba, Y., Nishmura, H., Nishiyama, T., Mizutani, J., 1987. Lichen acids, plant growth inhibitors from Usnea longissima. Phytochemistry 26, 3181–3185. Ogmundsdottir, H.M., Zoega, G.M., Gissurarson, S.R., Ingolfsdottir, K., 1998. Anti-proliferative effects of lichen-derived inhibitors of 5lipoxygenase on malignant cell-lines and mitogen-stimulated lymphocytes. Journal of Pharmacy and Pharmacology 50, 107–115.
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Effect of usnic acid from the lichen Cladonia substellata on Trypanosoma cruzi in vitro: an ultrastructural study E.A.B. De Carvalhoa, P.P. Andradeb, N.H. Silvac, E.C. Pereirad, R.C.B.Q. Figueiredoe,* a
Laborato´rio de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-901 Recife, PE, Brazil b Departamento de Gene´tica, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-901 Recife, PE, Brazil c Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-420 Recife, PE, Brazil d Departamento de Cieˆncias Geograficas; Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-901 Recife, PE, Brazil e Departamento de Biologia Celular e Ultraestrutura, Centro de Pesquisas Aggeu Magalha˜es/FIOCRUZ, Universidade Federal de Pernambuco, Av. Moraes Rego S/N8, 50670-420 Recife, PE, Brazil Received 7 June 2004; revised 16 September 2004; accepted 16 September 2004
Abstract Chemotherapy for Chagas’ disease is still unsatisfactory due to toxicity and limited effectiveness of the available drugs. In this work we have investigated the effect of usnic acid, isolated from lichen Cladonia substellata, against Trypanosoma cruzi, in vitro. Incubation of culture epimastigotes with 5–30 mg/ml of this compound resulted in growth inhibition in a dosis-dependent manner. Ultrastructural analysis of treated epimastigotes showed damage to mitochondria, with a marked increase in kinetoplast volume and vacuolation of the mitochondrial matrix. Intense lysis of bloodstream trypomastigotes was observed with all drug concentrations tested. Besides mitochondrial and kinetoplast damage, trypomastigotes also presented enlargement of the flagellar pocket, as well as intense cytoplasm vacuolation. Treatment of infected macrophages with 40 or 80 mg/ml usnic acid induced marked cytoplasm vacuolation in intracellular amastigote forms, with disorganization of parasite kinetoplast and mitochondria, but with no significant ultrastructural damage to the host cells. q 2004 Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Cladonia substellata; Usnic acid; Ultrastructure
1. Introduction Chagas’ disease is a serious health problem in South America, particularly in Brazil. More than 5 million Brazilians are affected, with major looses in work ability and life quality (WHO, 1990; Moncayo, 1993). Etiological agent of this disease is the haemoflagellate protozoan Trypanosoma cruzi, which is transmitted to humans and other mammals by triatomine bugs, with further human infection occurring by blood transfusion and transplacentally. Therapy for Chagas’ disease is unsatisfactory, due to the significant toxicity of the available drugs (nifurtimox and benznidazole). Although both compounds may reduce the acute phase and decrease mortality, parasitological cure is achieved in only 60% of acute patients and their use * Corresponding author. Tel.: C55 81 21012556; fax: C55 81 34532449. E-mail address: [email protected] (R.C.B.Q. Figueiredo). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.09.003
during the chronic phase of the disease is a matter of considerable discussion. (De Castro, 1993; Estani et al., 1998). Furthermore, differences in the susceptibility of T. cruzi strains to these drugs have been reported (Filardi et al., 1987). Lichens are symbiotic associations between fungal (mycobiont) and algal or cyanobacterial (photobiont) partners. They are able to synthesize several metabolites, comprising aliphatic, cycloaliphatic, aromatic and terpenic compounds (Huneck, 1999). Lichens have been used for medicinal purposes throughout the ages: some of them, as Cetraria islandica, Lobaria pulmonaria and Cladonia species, were reported to be effective in the treatment of pulmonary tuberculosis (Vartia, 1973). Usnic acid, [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)dibenzo-furandione; C18H16O7], is a yellow cortical pigment found only in lichens, occurring under two enantiomeric forms. Besides its antimicrobial activity
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against human and plant pathogens, usnic acid exhibits antiproliferative, anti-inflammatory and analgesic activities. (Al-Bekairi et al., 1991; Okuyama et al., 1995; Lauterwein et al., 1995; Ingolfsdottir et al., 1998; Ogmundsdottir et al., 1998). Previous studies have shown that usnic acid can cause the uncoupling of the oxidative phosphorilation of mitochondria (Abo-Khatwa et al., 1996), and thereby it has been used as a fat burner. On the other hand, deleterious effects of usnic acid on mammalian cells have been reported, by inhibiting cell proliferation and causing cytotoxicity in cultured keratinocytes (Kumar and Muller, 1999). Treatment of mice with usnic acid inhibited the proliferation of polychromatic erythrocytes (Al-Bekairi et al., 1991). Despite the potential use of usnic acid and other lichen compounds as chemotherapic drugs, few investigations have been performed on their activity against parasitic protozoa. It has been shown showed that (K)usnic acid exhibited a strong effect against Trichomonas vaginalis in vitro (Wu et al., 1995). Intralesional administration of usnic acid in BALB/c mice infected with promastigote forms of Leishmania, produced a significant reduction of cutaneous lesion (Fournet et al., 1997). The aim of this study was to analyze, at the ultrastructural level, the in vitro effects of usnic acid extracted from Cladonia substellata against the protozoan T. cruzi, the etiological agent of Chagas’ disease.
was spectrophotometrically estimated at 630 nm, in duplicate. Optical density was assumed to be directly correlated to parasite number in culture medium. Epimastigotes growing in LIT medium containing 1% DMSO were used as a control. Statistical analysis of growth differences between treated and control cultures was performed using the Wilcoxon Test, with p!0.05. After 5 days, control and treated parasites were harvested by centrifugation and processed for transmission electron microscopy, as described below.
2. Materials and methods
2.5. Effect of usnic acid on amastigotes
2.1. Parasites
Peritoneal macrophages were obtained from Swiss Webster mice as described elsewhere (Araujo-Jorge et al., 1989) Macrophage cultures were maintained with RPMI medium and then infected with bloodstream trypomastigotes at a parasite/cell ratio of 10:1. After 1 h, noninternalized parasites were washed out and fresh RPMI medium was added. Three days after infection, fresh medium with different concentrations of usnic acid (20, 40 and 80 mg/ml) was added and cells were cultivated for additional 24 h. At the end of the incubation time, control and treated–infected cells were fixed and processed for electron microscopy.
T. cruzi epimastigotes (Dm28c clone) were axenically maintained at 28 8C in LIT medium supplemented with 10% fetal bovine serum (Camargo, 1964) and harvested at the exponential phase of growth. Bloodstream trypomastigotes (Y strain) were obtained from infected albino Swiss mice at the peak of parasitemia, as described elsewhere (Meirelles et al., 1984). Intracellular amastigotes were obtained by infection of mouse peritoneal macrophages as described below.
2.4. Effect of usnic acid on bloodstream trypomastigotes Bloodstream trypomastigote forms were ressuspended in RPMI medium (Sigma Chemical Co., USA) supplemented with 2.5% fetal bovine serum and inoculated in 24-well plates at 4.3!105 cells/ml, in absence or presence of 5, 10, 20,40 and 80 mg/ml usnic acid. The parasites were cultivated for 72 h at 37 8C and the number of viable trypomastigotes/ml was estimated by daily counting using a haemocytometer. Counts were performed in triplicate and statistically analyzed with the Student’s t test, with p!0.005. For ultrastructural analysis, bloodstream trypomastigotes forms were ressuspended in RPMI medium, incubated in absence or presence of 20 mg/ml of usnic acid for 24 h at 37 8C and then processed for transmission electron microscopy.
2.2. Drugs 2.6. Transmission electron microscopy Usnic acid was isolated from the crude extract of the lichen C. substellata. Fractionation and purification of this compound were performed as previously described (Pereira et al., 1995). A stock solution was prepared at 1 mg/ml in 1% DMSO and kept at K20 8C until use. 2.3. Effect of usnic acid on epimastigotes Different drug concentrations (5, 10, 20, 30, 40, 50 mg/ml, diluted in LIT medium) were added to cell culture flasks containing 2!106 cells/ml. Aliquots (1 ml) were daily collected during five days and the culture growth
Control and treated culture epimastigotes, bloodstream trypomastigotes and infected macrophages were harvested, at room temperature by centrifugation at 1500g, washed twice in PBS and fixed for 2 h at 4 8C in a solution containing 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M cacodylate buffer, pH 7.2. After washing in this same buffer, cells were post-fixed for 1 h in a solution containing 1% OsO4, 0.8% potassium ferricyanide and 5 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.2. The cells were then dehydrated in acetone and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead
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citrate and examined in a Zeiss EM 109 transmission electron microscope.
3. Results Incubation of epimastigote cultures of T. cruzi with different concentrations of usnic acid resulted in a strong decrease of O.D. values, in a dosis-dependent manner. When 5 mg/ml of usnic acid were added to the medium, slight growth inhibition was observed only after 48 h of cultivation. However, the inhibitory effect on parasite proliferation was higher and statistically significant at concentrations ranging from 10 to 50 mg/ml. (Fig. 1). Changes in cell morphology, including increase in cell volume, loss of cell polarity due to nuclear and kinetoplast misplacement, and abnormally shaped body were observed in several parasites, as seen by light microscopy (data not shown). These effects were observed with all drug concentrations tested, but the number of affected cells and the severity of morphological changes depended on the drug concentration. Incubation of control epimastigotes with DMSO alone resulted in no significant effect on cell growth and morphology. Observation of untreated epimastigotes by transmission electron microscopy showed a centrally located nucleus, a single elongated mitochondrion running along the cell body, a compact condensation of mitochondrial kinetoplast DNA and reservosomes, the latter corresponding to the prelysosomal compartments in this parasite (Fig. 2). Ultrastructural analysis of epimastigotes treated with 5–10 mg/ml
Fig. 1. Inhibitory effects of usnic acid on epimastigote growth: (&) control; (%) 5 mg/ml; (C) 10 mg/ml; (:) 20 mg/ml; (*) 30 mg/ml; (,) 40 mg/ml and (B) 50 mg/ml. The points represent the mean value of two independent measurements.
Fig. 2. Ultrastructure of control epimastigote form grown in LIT medium. Longitudinal section showing reservosomes (R) at the posterior end, the centrally located nucleus (N) and rod-shaped kinetoplast (K). Mitochondrial profiles (arrow) are observed immediately beneath the plasma membrane. Bar: 0.5 mm.
usnic acid showed small changes in the cytoplasm density, cell swelling and loss of cell polarity (Fig. 3). Marked swelling and branching of mitochondria (Fig. 4), as well as loss of organization of the mitochondrial cristae, could be observed in cells treated with 30–50 mg/ml usnic acid. Highly condensed chromatin and severe vacuolation of mitochondria were usually seen at higher drug concentrations (Fig. 5). The Golgi apparatus, the endoplasmic
Fig. 3. Low magnification of epimastigote culture treated with 5 mg/ml of usnic acid showing eletron-luscent cytoplasm (*) and loss of cell polarity. Reservosomes (arrow) are spread throughout the cytoplasm. Bar: 5 mm.
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Fig. 4. Pronounced mitochondrial branching (m) can be observed throughout the cytoplasm of an epimastigote form incubated with 30 mg/ml usnic acid. Bar: 0.25 mm.
reticulum and the reservosomes remained essentially unaffected. Bloodstream trypomastigotes remained motile and with no visible changes in cell shape for the first 15 h in the presence of usnic acid, with all concentrations tested. However, 100% cell death was observed at 24 h for 40 and 80 mg/ml usnic acid, as well as at 48 and 72 h for drug
Fig. 5. Electron microscopy of epimastigote form treated with 50 mg/ml usnic acid showing marked vacuolation (arrow) of mitochondrial matrix and blebbing of mitochondrial membrane (arrowhead) Changes in the nucleus (N), including electron-luscent appearance of nuclear matrix and chromatin condensation can be observed. Bar: 0.25 mm.
Fig. 6. Effect of different concentrations of usnic acid on bloodstream tripomastigote viability in vitro. (C) 40 and 80 mg/ml, (!) 20 mg/ml, (:) 10 mg/ml, (&) 5 mg/ml, (%) controlCDMSO. Each point is the mean of three independent countings. All values are given as meanGSD. For all concentrations the effect of usnic acid on bloodstream trypomastigotes was statically significant (p!0.05).
concentrations of 20 and 10 mg/ml, respectively. A progressive decrease of trypomastigote viability was observed when parasites were incubated with 5 mg/ml usnic acid, reaching about 72% parasite death after 72 h of cultivation (Fig. 6). At the ultrastructural level, untreated bloodstream
Fig. 7. Effect of usnic acid treatment on bloodstream trypomastigotes of T. cruzi. Ultrastructure appearance of a control bloodstream trypomastigote (T) showing well preserved mitochondrion (arrow) and homogeneous cytoplasm.
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of the drug and changes induced by the drug treatment were investigated by both light and electron microscopy. In untreated macrophages, well preserved trypomastigotes and amastigotes could be observed in the host cell cytoplasm (Fig. 9a). No major alteration in this pattern was observed in infected cells treated with 20 mg usnic acid for 24 h. On the other hand, incubation with 40 and 80 mg/ml caused marked changes on the parasite ultrastructure. Amastigote forms exhibited mitochondrial and kinetoplast swelling and intense vacuolation (Fig. 9b), as also observed with the trypomastigote and epimastigote forms. An electron-luscent space with membrane profiles could be observed between the parasite and the host cell cytoplasm. At 80 mg/ml no intact intracellular parasites could be found (Fig. 9b), but only minor ultrastructural changes were detected in mitochondria, endoplasmic reticulum and other organelles of host cells. The number of infected cell dropped significantly when observed by light microscopy (data not shown).
Fig. 8. Effect of usnic acid treatment on bloodstream trypomastigotes of T. cruzi. Incubation with 20 mg/ml usnic acid induces an increase of mitochondrial (m) and kinetoplast (K) volume, loss of cytoplasmic content and vacuolation (*). Bar: 1 mm.
trypomastigotes presented a small flagellar pocket, an anterior flagellum, several electron-dense granules, a single mitochondrion (Fig. 7), and a characteristic basket-shaped kinetoplast. In treated trypomastigotes the major morphological changes were observed in the mitochondrion and kinetoplast (Fig. 8), although considerable swelling of the flagellar pocket and intense cytoplasmic vacuolation were also observed. Round shaped parasites were usually found. In order to analyze the ultrastructural effects of usnic acid on the clinically relevant intracellular amastigote forms, infected macrophages were treated with 20, 40 and 80 mg/ml
4. Discussion Usnic acid, which exists in nature in both (C) and (K) forms, is the most abundant constituent of several lichen species, including those belonging to the Usnea and Cladonia genera (Nishtoba et al., 1987) This compound is known to present several biological activities, acting as an anti-microbial, anti-tumoral and anti-inflammatory agent (Cocchietto et al., 2002). Our data demonstrate for the first time the effects of usnic acid on the protozoan T. cruzi. We initially tested the effect of several usnic acid concentrations on the epimastigotes proliferation in vitro. The strong decrease of O.D. values found in usnic acid treated cultures points to an effect on both growth and viability of epimastigote forms. The growth inhibition pattern was similar to that found when a crude extract of
Fig. 9. Effect of usnic acid on amastigotes. (a) Untreated macrophage showing well preserved parasites (arrow). Bar: 1 mm. (b) T. cruzi-infected macrophage treated with 80 mg/ml usnic acid for 24 h. Note the parasite disorganization, with swelling of the mitochondrion and the kinetoplast (*), as well as eletronluscent appearance of the nucleus (N) and chromatin condensation. Bar: 0.5 mm.
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C. substellata, containing about 90% usnic acid (Ahti et al., 1993; Pereira et al., 1994) was used (unpublished data). The morphological effects observed on mitochondria and kinetoplast suggest that the drug affects the energy metabolism of epimastigote. Indeed, it has been suggested that usnic acid acts as an uncoupler of oxidative phosphorylation in mouse liver mitochondria (Abo-Khatwa et al., 1996). Furthermore, the lipophylic (Muller, 2001) and ionization characteristics of this compound may alter the mitochondrial membranes, causing the organelle collapse. No significant difference in culture growth was detected at 5 mg/ml, when compared to the control. The weak effect observed at 5 mg/ml may be due to the existence of glycosomes in T. cruzi, which are organelles involved in the glycolytic pathway in trypanosomatids (Opperdoes and Michels, 1993). It is possible that at lower drug concentrations, glycosomes supply the deficiency in energy caused by the mitochondrial damage. Incubation of bloodstream trypomastigotes with usnic acid resulted in lysis of parasites, even at low drug concentrations. Although the ultrastructural observations suggest that mitochondrion and kinetoplast were the targets for usnic acid, as demonstrated for epimastigote forms, cytoplasmic vacuolation and swelling of the flagellar pocket were usually found in bloodstream trypomastigotes. These additional effects suggest that trypomastigotes are more susceptible to usnic acid treatment, probably due to differences in membrane permeability between the two forms (De Souza, 1999). Amastigotes were also affected by usnic acid treatment, although the drug concentration needed to achieve mitochondria and kinetoplast swelling were higher than those used for epimastigote and trypomastigote forms. Contrary to the high toxicity reported in the literature (Han et al., 2004; Pramyothin et al., 2004), the use of usnic acid at 80 mg/ml for 24 h was not toxic to murine peritoneal macrophages, since no significant alterations on cell morphology and ultrastructure could be observed under these conditions. Furthermore, no side effects were observed in T. cruzi-infected mice treated for 5 days with 25 mg/kg/day of usnic acid (unpublished data). Usnic acid has been shown to interfere with normal mitochondrial metabolism (Han et al., 2004), which may cause an increase in reactive oxygen species leading to both parasite and host cell death. However, the drug concentration needed to damage the host cell is considerably higher than those used in our experiments. T. cruzi is deficient in the detoxification mechanism of oxygen metabolites, and therefore is more sensitive to oxidative stress than vertebrate cells (Schirmer et al., 1987). In addition, the combination of lower drug concentrations with reduced time of treatment could help to solve this question. Furthermore, usnic acid incorporation into nanoparticles of biocompatible polymers is currently underway in our laboratory in an attempt to overcome the deleterious effect of this compound on the host cells.
In conclusion, although further studies are needed in order to elucidate the pathophysiological effects of usnic acid, either in vivo or in vitro, our results point towards the potential use of usnic acid as a chemotherapic agent for Chagas’ disease.
Acknowledgements The authors would like to thank Mr Raimundo Nazareno C. Pimentel for his technical assistance. This work was supported by CPqAM/FIOCRUZ, and UFPE.
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