3b), (3) wrinkled cells (Figs 3c and 4a–c), and (4) the formation of cell clusters (Fig. 4a). Quantitative analyses revealed that membrane disruption and wrinkled cells were the most common alterations observed (Fig. 5). A small increase of rounded cells (Figs 3d and 4a–c) percentage was observed in T. vaginalis treated with AZA whereas fewer of these cells were found after EIL treatment (Fig. 5). However, no change was observed on endoflagellar forms (pseudocysts). These cells appear under unfavourable environmental conditions when the flagella are internalized, and a true cell wall is not formed (Pereira-Neves et al., 2003). In addition, changes LBH589 in intracellular structures

were also observed. Several important alterations were observed at the ultrastructural level in the drug-treated trophozoites, including (1) Golgi duplication, (2) vesicles containing membranous Luminespib supplier profiles (Fig. 4d), (Fig. 6a–c), (3) altered and enlarged Golgi cisternae (Fig. 4e, arrow) and (4) abnormal hydrogenosomes, which are seen as electron-lucent organelles (Fig. 4d–f). Autophagy was also detected, as evidenced by the membranous profiles of endoplasmic reticulum that were surrounding the hydrogenosomes (Fig. 4f,

arrowhead). Giardia lamblia treated with AZA and EIL also presented similar results (Maia et al., 2007) and this autophagic process suggests that the cells were implementing a survival strategy under stress conditions (Edinger & Thompson, 2004). Many of these alterations have previously been shown in T. vaginalis treated with hydrogen peroxide (Mariante et al., 2003), taxol (Madeiro

da Costa & Benchimol, 2004), nocodazole (Madeiro da Costa & Benchimol, 2004) or griseofulvin (Mariante et al., 2006). It is important to note that the phenomena described above, such as membrane blebbing, vacuolization and autophagy, are features typical of cell death, and they have been described previously in trichomonads (Mariante et al., 2006; Benchimol, 2008). Moreover, the presence of autophagic vacuoles may be indicative of membrane recycling, thus aiding in the remodelling of the cell (Maia et al., 2007). However, in previous studies of T. vaginalis treated with metronidazole, the main alteration observed was a reduction in hydrogenosomes’ Amine dehydrogenase size (Land et al., 2001; Wright et al., 2010). On the other hand, in mammalian cells (MDCK cells) where the enzyme target is also absent, no morphology alteration was observed by SEM and TEM (Supporting Information, Figs S1 and S2). Mammalian cells, such as MDCK and Caco cells, did not exhibit any apparent damage when treated with 5 μM AZA or 10 μM EIL for 24 h and analysed using the MTT viability test (Fig. S3). This is a very important observation, as it suggests that the experimental compounds have selective antiparasitic effects. Taken together, the results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.