Trypanosoma cruzi mitochondrial swelling and membrane potential collapse as primary evidence of the mode of action of naphthoquinone analogues

Abstract

BackgroundNaphthoquinones (NQs) are privileged structures in medicinal chemistry due to the biological effects associated with the induction of oxidative stress. The present study evaluated the activities of sixteen NQs derivatives on Trypanosoma cruzi.ResultsFourteen NQs displayed higher activity against bloodstream trypomastigotes of T. cruzi than benznidazole. Further assays with NQ1, NQ8, NQ9 and NQ12 showed inhibition of the proliferation of axenic epimastigotes and intracelulluar amastigotes interiorized in macrophages and in heart muscle cells. NQ8 was the most active NQ against both proliferative forms of T. cruzi. In epimastigotes the four NQs induced mitochondrial swelling, vacuolization, and flagellar blebbing. The treatment with NQs also induced the appearance of large endoplasmic reticulum profiles surrounding different cellular structures and of myelin-like membranous contours, morphological characteristics of an autophagic process. At IC50 concentration, NQ8 totally disrupted the ΔΨm of about 20% of the parasites, suggesting the induction of a sub-population with metabolically inactive mitochondria. On the other hand, NQ1, NQ9 or NQ12 led only to a discrete decrease of TMRE + labeling at IC50 values. NQ8 led also to an increase in the percentage of parasites labeled with DHE, indicative of ROS production, possibly the cause of the observed mitochondrial swelling. The other three NQs behaved similarly to untreated controls.ConclusionsNQ1, NQ8, NQ9 and NQ12 induce an autophagic phenotype in T. cruzi epimastigoted, as already observed with others NQs. The absence of oxidative stress in NQ1-, NQ9- and NQ12-treated parasites could be due to the existence of more than one mechanism of action involved in their trypanocidal activity, leaving ROS generation suppressed by the detoxification system of the parasite. The strong redox effect of NQ8 could be associated to the presence of the acetyl group in its structure facilitating quinone reduction, as previously demonstrated by electrochemical analysis. Further experiments using biochemical and molecular approaches are needed to better characterize ROS participation in the mechanism of action of these NQs.

Phytotoxic action of naphthoquinone juglone demonstrated on lettuce seedling roots

“…However, there is only limited evidence about changes in the structure of endoplasmic reticulum under treatment with juglone. Salomao et al, who investigated effect of naphthoquinone analogues on Trypanosoma cruzii, observed mitochondrial swelling and membrane potential collapse as well as large endoplasmic reticulum profiles surrounding different cellular structures (Salomao et al, 2013). Finally, we observed DNA fragmentation, which was revealed using two methods, the TUNEL test and DNA electrophoresis.…”

Effects of a novel β–lapachone derivative on Trypanosoma cruzi : Parasite death involving apoptosis, autophagy and necrosis

“…The lapachone-induced mitochondrial dilation and autophagy (mitophagy) mechanisms depend on the production of ROS (Salomão et al., 2013). The R72-treated parasites not only removed mitochondrial portions by mitophagy, but also showed mitochondria in the condensed configuration with distended cristae, long known to be metabolically inactive (Hackenbrock et al., 1971).…”

“…Hence, mitophagy and mitochondrial loss of function may lead to metabolic deficit, possibly promoting parasite necrosis. β-lapachone and its derivatives were shown to trigger apoptosis and autophagy in both parasites (Docampo et al., 1978, Menna-Barreto et al., 2009a, Salomão et al., 2013) and tumor cells (Li et al., 1999, Li et al, 2003; Park et al., 2011, Di Rosso et al., 2013), involving the formation of ROS (Araújo et al., 2012, Salomão et al., 2013). …”

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

“…For transmission electron microscopy observation, cells over coverslips were fixed and post-fixed after 48 h of drug treatment as described above, dehydrated in an ascending acetone series and embedded in PolyBed 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in Jeol JEM1011 transmission electron microscope (Tokyo, Japan) at Plataforma de Microscopia Eletrônica, IOC, FIOCRUZ (Salomão et al 2013). …”