Leishmaniasis are infectious diseases caused by protozoa of the genus Leishmania, that are digenetic parasites alternating between an extracellular promastigote stage (in sand fly vector) and an intracellular amastigote stage (in mammalian host). Promastigote phagocytosis results in a macrophage respiratory burst in vitro, leading to the generation of reactive oxygen species (ROS) such as superoxide anion (O2
-), hydrogen peroxide (H2O2), singlete oxygen (1O2) and hydroxyl radical (OH). Thus, we aimed to evaluate in vitro ROS toxicity for L. L. chagasi promastigotes, as well as for their amastigote
counterparts. To evaluate ROS toxicity for promastigotes, we selected 16 L. L. chagasi isolates (in log phase growth) and incubated them under increasing concentrations of menadione (0-750ìM) for 4 hours, after which we determined ROS viability for these parasites by quantifying the number of mobile forms. For ROS toxicity for amastigote L. L. chagasi, we infected J774.16, a murine macrophage cell line, with ROS susceptible and resistant L. L. chagasi (in stationary phase of growth) in cultures treated
by LMNA, an iNOS inhibitor (to block the synthesis of nitric oxide), treatment with diethyldithiocarbamate (DETC), a SOD-1 inhibitor (to increase superoxide anion synthesis) as well as incubation with N-acetylcysteine, NAC ( an antioxidant).These infection experiments were conducted in 8 well plates in a 5:1 ratio (parasites/cell). Subsequently, we incubated these plates for 4, 24, 48 and 72 hours at 37oC and 5% CO2. After that, the plates were stained and the number of amastigotes (parasite burden)
determined. We observed that 14 out 16 isolates treated with menadione showed 50% or more loss of their viability at concentrations varying from 15 to 750 ìM, whereas two of them exhibited even 70% or more viability at 750 mM. From ROS toxicity evaluation for L. L. chagasi amastigotes, we observed in J774.16 cultures infected by ROS susceptible and resistant L. L. chagasi a decrease in parasitic load for both isolates. This decrease in parasite burden was observed also in cultures treated by LMNA, an
iNOS inhibitor. Inhibition of SOD by DETC also promoted a decrease in the number of amastigotes for both of these J774.16 cultures from 24 hour infection. The addition of NAC to these cultures increased the number of amastigotes for ROS resistant infected J774.16 cells but nor for those ROS susceptible infected cultures. These data indicate that damage on these amastigotes induced by DETC is oxidative. Thus, it is possible to conclude that reactive oxygen species are crucial toxic agents for L. L. chagasi as in promastigote form, as well as in amastigote form. / As leishmanioses sao doencas infecciosas causadas por parasitos do genero Leishmania. Leishmania sao protozoarios digeneticos, alternando entre as formas promastigota (flebotomineo) e amastigota (hospedeiro mamifero). A fagocitose de promastigotas desencadeia em macrofagos um gburst oxidativo h, gerando especies reativas do oxigenio (ROS) como o anion superoxido (O2
-), peroxido de hidrogenio (H202), o oxigenio singlete (1O2) e o radical hidroxila (OH). Assim, objetivamos avaliar in vitro a atividade leishmanicida de ROS para promastigotas de L.(L.) chagasi, bem como para
suas respectivas formas amastigotas. Para a avaliacao da toxicidade de ROS para promastigotas, selecionamos 16 isolados de L.(L.) chagasi (em fase log de crescimento), incubando-os sob concentracoes crescentes de menadiona (0-750 ÊM) por um periodo de 4h, ao fim do qual determinamos a viabilidade desses parasitos, quantificando o numero de formas moveis. Na avaliacao da acao leishmanicida de ROS para as formas amastigotas de L. L. chagasi, infectamos uma linhagem celular de macrofagos murinos J774.16 com um isolado resistente e dois isolados susceptiveis a ROS (na proporcao 5:1 parasitos/celulas) em culturas com LMNA, inibidor de iNOS, (para bloquear a
sintese de oxido nitrico), incubadas com o inibidor da enzima SOD-1, dietilditiocarbamato, DETC (para aumentar a producao de O2 -) e com N-acetilcisteina, NAC (um antioxidante) em placas de 8 pocos e incubadas por 4, 24, 48 e 72 horas. Ao termino de cada incubacao, coramos as placas com panotipo, para determinacao do numero de amastigotas (carga parasitaria). A partir da exposicao de promastigotas a concentracoes crescentes de menadiona, observamos que dos 16 isolados avaliados, 14 deles apresentaram perdas de 50% ou mais de suas viabilidades entre concentracoes de 15 a 750 ÊM de menadiona (formas susceptiveis a ROS), enquanto apenas dois deles apresentaram 70% ou mais de viabilidade a 750 ÊM de menadiona (formas resistentes a ROS). Na avaliacao da toxicidade de ROS para as formas amastigotas, observamos nas culturas de celulas J774.16 infectadas por L. L. chagasi susceptiveis e resistente a ROS diminuicao na carga parasitaria de ambos os isolados a partir do periodo de 48 horas. Para as culturas incubadas com DETC, observamos a reducao dos numeros de
amastigotas para essas culturas, a partir do tempo de 24 horas de infeccao. A adicao de NAC a essas culturas reverteu a carga parasitaria das culturas infectadas pelo isolado resistente, mas nao as que foram infectadas pelos isolados susceptiveis a ROS, indicando que o dano induzido por DETC sobre essas amastigotas e oxidativo. Assim, e possivel afirmar que as especies reativas do oxigenio sao importantes agentes toxicos para promastigotas e amastigotas de L. L. chagasi.
| Identifer | oai:union.ndltd.org:IBICT/oai:ri.ufs.br:riufs/3258 |
| Date | 12 June 2013 |
| Creators | Carvalho, Sueli Silva de |
| Contributors | Leopoldo, Paulo de Tarso Goncalves |
| Publisher | Pós-Graduação em Biologia Parasitária |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFS, instname:Universidade Federal de Sergipe, instacron:UFS |
| Rights | info:eu-repo/semantics/openAccess |
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