Danielle Lazarin-Bidóia

Trypanocidal Action of (−)-Elatol Involves an Oxidative Stress Triggered by Mitochondria Dysfunction

Natural compounds have shown good potential for the discovery of new chemotherapeutics for the treatment of Chagas’ disease. Recently, our group reported the effective trypanocidal activity of (−)-elatol, extracted from the red macroalgae Laurencia dendroidea present in the Brazilian coast against Trypanosoma cruzi. However, the mechanism of action of this compound has remained unclear. There are only hypotheses concerning its action on mitochondrial function. Here, we further investigated the mechanisms of action of (−)-elatol on trypomastigotes of T. cruzi. For this, we evaluated some biochemical alterations in trypomastigotes treated with (−)-elatol. Our results show that (−)-elatol induced depolarization of the mitochondrial membrane, an increase in the formation of mitochondrial superoxide anion and loss of cell membrane and DNA integrity. Additionally, (−)-elatol induced formation of autophagic vacuoles and a decrease in cell volume. All together, these results suggest that the trypanocidal action of (−)-elatol involves multiple events and mitochondria might be the initial target organelle. Our hypothesis is that the mitochondrial dysfunction leads to an increase of ROS production through the electron transport chain, which affects cell membrane and DNA integrity leading to different types of parasite death.

Further evidence of the trypanocidal action of eupomatenoid-5: Confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi

Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC₅₀ 40.5 μM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.

Eupomatenoid-5 Isolated from Leaves of Piper regnellii Induces Apoptosis in Leishmania amazonensis

Leishmania spp. are protozoa responsible for leishmaniasis, a neglected disease that kills up to 50,000 people every year. Current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy and safety and increased drug resistance. An urgent need exists to find new and more affordable drugs. Our previous study demonstrated the antileishmanial activity of eupomatenoid-5, a neolignan obtained from leaves of Piper regnellii var. pallescens. The aim of the present study was to clarify the mode of action of eupomatenoid-5 against L. amazonensis. We used biochemical and morphological techniques and demonstrated that eupomatenoid-5 induced cell death in L. amazonensis promastigotes, sharing some phenotypic features observed in metazoan apoptosis, including increased reactive oxygen species production, hypopolarization of mitochondrial potential, phosphatidylserine exposure, decreased cell volume, and G0/G1 phase cell cycle arrest.

Unsymmetrical 1,5-diaryl-3-oxo-1,4-pentadienyls and their evaluation as antiparasitic agents.

In this work the synthesis and antiparasitical activity of new 1,5-diaryl-3-oxo-1,4-pentadienyl derivatives are described. First, compounds 1a, 1b, 1c and 1d were prepared by acid-catalyzed aldol reaction between 2-butanone and benzaldehyde, anisaldehyde, p-N,N-dimethylaminobenzaldehyde and p-nitrobenzaldehyde. Reacting each of the methyl ketones 1a, 1b, 1c and 1d with the p-substituted benzaldehydes under basic-catalyzed aldol reaction, we further prepared compounds 2a-2p. All twenty compounds were evaluated for antiproliferative activity, particularly for promastigote of Leishmania amazonensis and epimastigote of Trypanosoma cruzi. All compounds showed good activity while nitro compounds 2i and 2k showed inhibition activity at a few μM.

Polyelectrolyte complexes based on alginate/tanfloc: Optimization, characterization and medical application

Hydrogels based on alginate and tanfloc (a cationic biopolymer obtained from natural condensed tannins) were successfully prepared. Tanfloc (TN) presents high aqueous solubility at pHs lower than 10; it contains substituted amino sites and molar weight of ca. 600,000gmol-1. A factorial design (22) was used to optimize the yield of alginate/tanfloc polyelectrolyte complexes (PECs). Dialysis recovered the overplus of alginate (AG) no complexed with TN. These materials were characterized by thermal analyses (TGA/DTG and DSC), zeta potential, and FTIR, while SEM technique depicted a rough surface on AG/TN complex, containing non-homogeneous pores. Indeed, the AG and TN were tailored to elicit scaffold materials with outstanding cytocompatibility, mainly upon mouse preosteoblastic cells because of reconstruction of bone tissues (119% at 10days). The AG/TN complex also displayed antioxidant and bactericidal activities against Staphylococcus aureus (S. aureus). Besides, the pristine TN fostered bacteriostatic and bactericidal performances towards S. aureus and Escherichia coli. However, for our best knowledge, no studies were still carried out on TN and TN-based materials for medical purpose.

Scaffolds based on chitosan/pectin thermosensitive hydrogels containing gold nanoparticles

Thermosensitive hydrogels based on chitosan/pectin (CS/Pec) and CS/Pec/gold nanoparticles (CS/Pec/AuNPs) were successfully prepared with different AuNP levels. Using a tilting method, gelation temperature was demonstrated to decrease when the amount of AuNPs increased and pectin concentrations decreased. The presence of AuNPs in the CS/Pec composite was evaluated via WAXS and UV-vis techniques, while SEM analysis assessed the average size of pores (350-600μm). All samples were extremely cytocompatible with many cell types, such as normal kidney epithelial cells (VERO cells), epithelial colorectal adenocarcinoma cells (HT-29 cells), HPV-16 positive human cervical tumour cells (SiHa cells), kidney epithelial cells (LLCMK2 cells) and murine macrophage cells (J774A1 cells). Cell viability assays using the MTT method upon mouse preosteoblastic cells (MC3T3-E1 cells) showed that CS/Pec and CS/Pec/AuNPs composites had the potential to foster proliferation and growth of bone cells, making them possible stimulators for reconstruction of bone tissues.

The Combination of Vitamin K3 and Vitamin C Has Synergic Activity against Forms of Trypanosoma cruzi through a Redox Imbalance Process

Chagas’ disease is an infection that is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. Because of severe side effects and variable efficacy, the current treatments for Chagas’ disease are unsatisfactory, making the search for new chemotherapeutic agents essential. Previous studies have reported various biological activities of naphthoquinones, such as the trypanocidal and antitumor activity of vitamin K3. The combination of this vitamin with vitamin C exerted better effects against various cancer cells than when used alone. These effects have been attributed to an increase in reactive oxygen species generation. In the present study, we evaluated the activity of vitamin K3 and vitamin C, alone and in combination, against T. cruzi. The vitamin K3 + vitamin C combination exerted synergistic effects against three forms of T. cruzi, leading to morphological, ultrastructural, and functional changes by producing reactive species, decreasing reduced thiol groups, altering the cell cycle, causing lipid peroxidation, and forming autophagic vacuoles. Our hypothesis is that the vitamin K3 + vitamin C combination induces oxidative imbalance in T. cruzi, probably started by a redox cycling process that leads to parasite cell death.

Additional Evidence of the Trypanocidal Action of (−)-Elatol on Amastigote Forms through the Involvement of Reactive Oxygen Species

Chagas’ disease, a vector-transmitted infectious disease, is caused by the protozoa parasite Trypanosoma cruzi. Drugs that are currently available for the treatment of this disease are unsatisfactory, making the search for new chemotherapeutic agents a priority. We recently described the trypanocidal action of (−)-elatol, extracted from the macroalga Laurencia dendroidea. However, nothing has been described about the mechanism of action of this compound on amastigotes that are involved in the chronic phase of Chagas’ disease. The goal of the present study was to evaluate the effect of (−)-elatol on the formation of superoxide anions (O2•−), DNA fragmentation, and autophagy in amastigotes of T. cruzi to elucidate the possible mechanism of the trypanocidal action of (−)-elatol. Treatment of the amastigotes with (−)-elatol increased the formation of O2•− at all concentrations of (−)-elatol assayed compared with untreated parasites. Increased fluorescence was observed in parasites treated with (−)-elatol, indicating DNA fragmentation and the formation of autophagic compartments. The results suggest that the trypanocidal action of (−)-elatol might involve the induction of the autophagic and apoptotic death pathways triggered by an imbalance of the parasite’s redox metabolism.

Synthesis and evaluation of the trypanocidal activity of a series of 1,3,4-thiadiazoles derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones

In this study, we synthesized a series of 1,3,4-thiadiazole derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones (2a–k). We also determined the cytotoxicity in LLCMK2 cells and the activity against epimastigote and trypomastigote forms of Trypanosoma cruzi, of these synthetic compounds and also of a series of 1,3,4-thiadiazole without the monoterpene R-(+)-limonene (4a–k). 1,3,4-Thiadiazole compounds showed significant trypanocidal activity and a high selectivity indexes. The vast majority of the monoterpene derivatives, substituted by R-(+)-limonene, presented better anti-T. cruzi activity than the non-substituted compounds. Regarding the cytotoxic profile, the compounds without the monoterpene R-(+)-limonene were, in general, less toxic. The present findings indicate that the 1,3,4-thiadiazoles derivatives of R-limonene have potential trypanocidal activity that justify further studies to better understand the mechanism of action of these substances on T. cruzi.