Effect of usnic acid from the lichen Cladonia substellata on Trypanosoma cruzi in vitro: an ultrastructural study E.A.B. De Carvalho a , P.P. Andrade b , N.H. Silva c , E.C. Pereira d , R.C.B.Q. Figueiredo e, * 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 Cie ˆncias 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 transplacen- tally. 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 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; C 18 H 16 O 7 ], is a yellow cortical pigment found only in lichens, occurring under two enantiomeric forms. Besides its antimicrobial activity 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.09.003 Micron 36 (2005) 155–161 www.elsevier.com/locate/micron * Corresponding author. Tel.: C55 81 21012556; fax: C55 81 34532449. E-mail address: bressan@cpqam.fiocruz.br (R.C.B.Q. Figueiredo).