Characterization of the Effect of N-(2-Methoxyphenyl)-1-methyl-1H-benzimidazol-2-amine, Compound 8, against Leishmania mexicana and Its In Vivo Leishmanicidal Activity.

Chemotherapy currently available for leishmaniasis treatment has many adverse side effects and drug resistance. Therefore, the identification of new targets and the development of new drugs are urgently needed. Previously, we reported the synthesis of a N-(2-methoxyphenyl)-1-methyl-1H-benzimidazol-2-amine, named compound 8, with an IC50 value in the micromolar range against L. mexicana, it also inhibited 68.27% the activity of recombinant L. mexicana arginase. Herein, we report studies carried out to characterize the mechanism of action of compound 8, as well as its in vivo leishmanicidal activity. It was shown in our ultrastructural studies that compound 8 induces several changes, such as membrane blebbing, the presence of autophagosomes, membrane detachment and mitochondrial and kinetoplast disorganization, among others. Compound 8 triggers the production of ROS and parasite apoptosis. It reduced 71% of the parasite load of L. mexicana in an experimental model of cutaneous leishmaniasis in comparison with a control. Altogether, the data obtained suggest the potential use of compound 8 in the treatment of cutaneous leishmaniasis.

NAC Pre-Administration Prevents Cardiac Mitochondrial Bioenergetics, Dynamics, Biogenesis, and Redox Alteration in Folic Acid-AKI-Induced Cardio-Renal Syndrome Type 3.

The incidence of kidney disease is increasing worldwide. Acute kidney injury (AKI) can strongly favor cardio-renal syndrome (CRS) type 3 development. However, the mechanism involved in CRS development is not entirely understood. In this sense, mitochondrial impairment in both organs has become a central axis in CRS physiopathology. This study aimed to elucidate the molecular mechanisms associated with cardiac mitochondrial impairment and its role in CRS development in the folic acid-induced AKI (FA-AKI) model. Our results showed that 48 h after FA-AKI, the administration of N-acetyl-cysteine (NAC), a mitochondrial glutathione regulator, prevented the early increase in inflammatory and cell death markers and oxidative stress in the heart. This was associated with the ability of NAC to protect heart mitochondrial bioenergetics, principally oxidative phosphorylation (OXPHOS) and membrane potential, through complex I activity and the preservation of glutathione balance, thus preventing mitochondrial dynamics shifting to fission and the decreases in mitochondrial biogenesis and mass. Our data show, for the first time, that mitochondrial bioenergetics impairment plays a critical role in the mechanism that leads to heart damage. Furthermore, NAC heart mitochondrial preservation during an AKI event can be a valuable strategy to prevent CRS type 3 development.

Alcohol: Immunomodulatory Effects and Cancer.

Alcohol consumption has been linked to numerous pathologic conditions, including infectious diseases and several types of cancer. Alcohol exerts its modulatory effects on the immune system (IS) in a dose- and time-dependent manner. Numerous studies indicate that these alterations affect responses such as peripheral inflammation or decreased antibody production and promote chronic inflammation, leading to cell death. The molecular mechanisms underlying these effects involve generating an oxidative tissue environment, producing cell damage-associated molecular patterns (DAMPs), and activating pattern recognition receptors. In particular, toll-like receptors and their signaling system emerge as central elements whose activity is altered by alcohol intake. There is also some epidemiological evidence demonstrating the causal role of alcohol in the development of various types of cancer, such as head-and-neck cancer, esophageal cancer, colorectal cancer, liver cancer, and breast cancer. Most recent evidence suggests that factors related to alcohol consumption and cancer include increased levels of acetaldehyde, production of reactive oxygen species, alteration in DNA methylation, and modifications in retinoid metabolism. In addition, changes associated with alcohol use on the IS and intestinal microbiota may favor the growth of some types of tumors.

Alcohol: Immunomodulatory Effects and Cancer

ABSTRACT Alcohol consumption has been linked to numerous pathologic conditions, including infectious diseases and several types of cancer. Alcohol exerts its modulatory effects on the immune system (IS) in a dose- and time-dependent manner. Numerous studies indicate that these alterations affect responses such as peripheral inflammation or decreased antibody production and promote chronic inflammation, leading to cell death. The molecular mechanisms underlying these effects involve generating an oxidative tissue environment, producing cell damage-associated molecular patterns (DAMPs), and activating pattern recognition receptors. In particular, toll-like receptors and their signaling system emerge as central elements whose activity is altered by alcohol intake. There is also some epidemiological evidence demonstrating the causal role of alcohol in the development of various types of cancer, such as head-and-neck cancer, esophageal cancer, colorectal cancer, liver cancer, and breast cancer. Most recent evidence suggests that factors related to alcohol consumption and cancer include increased levels of acetaldehyde, production of reactive oxygen species, alteration in DNA methylation, and modifications in retinoid metabolism. In addition, changes associated with alcohol use on the IS and intestinal microbiota may favor the growth of some types of tumors.

Calpain Regulates Reactive Oxygen Species Production during Capacitation through the Activation of NOX2 and NOX4.

Capacitation is a series of physiological, biochemical, and metabolic changes experienced by mammalian spermatozoa. These changes enable them to fertilize eggs. The capacitation prepares the spermatozoa to undergo the acrosomal reaction and hyperactivated motility. Several mechanisms that regulate capacitation are known, although they have not been fully disclosed; among them, reactive oxygen species (ROS) play an essential role in the normal development of capacitation. NADPH oxidases (NOXs) are a family of enzymes responsible for ROS production. Although their presence in mammalian sperm is known, little is known about their participation in sperm physiology. This work aimed to identify the NOXs related to the production of ROS in guinea pig and mouse spermatozoa and define their participation in capacitation, acrosomal reaction, and motility. Additionally, a mechanism for NOXs' activation during capacitation was established. The results show that guinea pig and mouse spermatozoa express NOX2 and NOX4, which initiate ROS production during capacitation. NOXs inhibition by VAS2870 led to an early increase in the capacitation and intracellular concentration of Ca2+ in such a way that the spermatozoa also presented an early acrosome reaction. In addition, the inhibition of NOX2 and NOX4 reduced progressive motility and hyperactive motility. NOX2 and NOX4 were found to interact with each other prior to capacitation. This interaction was interrupted during capacitation and correlated with the increase in ROS. Interestingly, the association between NOX2-NOX4 and their activation depends on calpain activation, since the inhibition of this Ca2+-dependent protease prevents NOX2-NOX4 from dissociating and ROS production. The results indicate that NOX2 and NOX4 could be the most important ROS producers during guinea pig and mouse sperm capacitation and that their activation depends on calpain.

Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production.

The F1FO-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-ß-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.

Liposomes Containing Amaranth Unsaponifiable Matter and Soybean Lunasin Suppress ROS Production in Fibroblasts and Reduced Interleukin Production in Macrophages.

Inflammation is a normal response in defense to agents that may cause damage to the human body. When inflammation becomes chronic, reactive oxygen species (ROS) are produced; which could lead to diseases such as cancer. The aim was to assess liposomes' antioxidant and anti-inflammatory capacity loaded with amaranth unsaponifiable matter and soybean lunasin (UM + LunLip) in an in vitro model using fibroblasts and macrophages. To evaluate ROS production, fibroblasts CHON-002 ABAP were added to promote ROS production; and the cells were treated with UM + LunLip. For inflammation markers production, lipopolysaccharides (LPS)-stimulated RAW 264.7 and peritoneal macrophages were treated with empty liposomes (EmLip), liposomes loaded with unsaponifiable matter (UMLip), liposomes loaded with lunasin (LunLip), and UM + LunLip. ROS production was significantly decreased by 77% (p < 0.05) when fibroblasts were treated with UM + LunLip at 2 mg lunasin/mL compared with the control treated with ABAP. Treatment with UMLip was the most effective in reducing tumor necrosis factor-α (71-90%) and interleukin-6 (43-55%, p < 0.001). Both liposomes containing unsaponifiable matter (UMLip and UM + LunLip) were more effective than EmLip or LunLip. In conclusion, amaranth unsaponifiable matter-loaded liposomes are effective in decreasing pro-inflammatory cytokine production.

Linearolactone Induces Necrotic-like Death in Giardia intestinalis Trophozoites: Prediction of a Likely Target.

Linearolactone (LL) is a neo-clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on Giardia intestinalis trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand-protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from G. intestinalis. LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.

Antiproliferative activity of two copper (II) complexes on colorectal cancer cell models: Impact on ROS production, apoptosis induction and NF-κB inhibition.

The main goal of this work was to screen the antiproliferative activity and mechanism of actions of two copper complexes: [Cu(dmp)2(CH3CN)]2+ (1) and [Cu(phen)2(CH3CN)]2+ (2) on 2D and 3D colorectal cancer cells models. Cell viability studies on three colorectal cancer cell lines (HT-29, LS174T, Caco-2) displayed that 1 showed more robust antiproliferative activity than 2 and cisplatin. Intracellular copper content (63.24% and 48.06% for 1 and 2, respectively) can explain the differences in the cytotoxicity assay. ROS production is the primary mechanism of action involved in the antiproliferative activity of 1 showing 4-, 70- and 2.5- fold increased values of ROS level for HT-29, LS174T, Caco-2 cancer cell lines, respectively. This effect takes place along with the depolarization of the mitochondrial membrane at 2 µM. Besides, both complexes increased apoptosis on three cancer cell lines at low micromolar concentrations (0.5-2.5 µM). Moreover, 1 and 2 inhibited NF-κB pathway both in HT-29-NF-kB-hrGFP monolayer (0.5 to 1 µM) and spheroids HT-29 GFP (5 to 10 µM). This inhibitory effect leads to an inactivation of the MMP-9 expression on HT-29 cell line. Altogether, these results showed that 1 exhibits antiproliferative activity on human colorectal cancer cells in the monolayer and the 3D model.

Incomptine A Induces Apoptosis, ROS Production and a Differential Protein Expression on Non-Hodgkin's Lymphoma Cells.

Sesquiterpene lactones are of pharmaceutical interest due their cytotoxic and antitumor properties, which are commonly found within plants of several genera from the Asteraceae family such as the Decachaeta genus. From Decachaeta incompta four heliangolide, namely incomptines A-D have been isolated. In this study, cytotoxic properties of incomptine A (IA) were evaluated on four lymphoma cancer cell lines: U-937, Farage, SU-DHL-2, and REC-1. The type of cell death induced by IA and its effects on U-937 cells were analyzed based on its capability to induce apoptosis and produce reactive oxygen species (ROS) through flow cytometry with 4',6-diamidino-2-phenylindole staining, dual annexin V/DAPI staining, and dichlorofluorescein 2',7'-diacetate, respectively. A differential protein expression analysis study was carried out by isobaric tags for relative and absolute quantitation (iTRAQ) through UPLC-MS/MS. Results reveal that IA exhibited cytotoxic activity against the cell line U-937 (CC50 of 0.12 ± 0.02 µM) and the incubation of these cells in presence of IA significantly increased apoptotic population and intracellular ROS levels. In the proteomic approach 1548 proteins were differentially expressed, out of which 587 exhibited a fold-change ≥ 1.5 and 961 a fold-change ≤ 0.67. Most of these differentially regulated proteins are involved in apoptosis, oxidative stress, glycolytic metabolism, or cytoskeleton structuration.

Sildenafil attenuates nonsteroidal anti-inflammatory-induced gastric ulceration in mice via antioxidant and antigenotoxic mechanisms.

Sildenafil (SIL) has potential as an interesting gastroprotective drug. However, the pathways of its protective effect still needs to be clarified, and its use as a potential gastroprotective agent validated. This study aims to evaluate the effects of SIL via modulation of oxidative stress in a NSAID-induced gastric lesion model. Male Swiss mice were divided into six groups: control (CON, water), nonsteroidal anti-inflammatory drug (NSAID, water), proton pump inhibitor (PPI, 30 mg/kg of lansoprazole), SIL 5 (5 mg/kg), SIL 25 (25 mg/kg) and SIL 50 (50 mg/kg). The animals were treated by gavage (a single dose) after 24 hours of fasting, and gastric lesions were performed after 30 minutes, with indomethacin (40 mg/kg, by gavage). After 6h, the animals were killed and the stomach was removed to evaluate reactive oxygen species (ROS) production, oxidation of macromolecules, quantification of antioxidant enzymes, DNA fragmentation, apoptosis and macroscopic and histologic analysis of gastric lesions. SIL exerts a dose-dependent gastroprotective effect against NSAID-induced mucosal injury, also reducing cytoplasmic levels of ROS and consequent oxidative damage to macromolecules. In addition, SIL increases nitric oxide bioavailability, antioxidant enzymes and gastric cellular viability, as well as restoring important factors involved in gastroprotection. Our results demonstrate that different doses of SIL prevent indomethacin-induced gastric ulcer in mice via different, but complementary antioxidant, antigenotoxic and antiapoptotic mechanisms.

The Effect of Rutin and Extracts of Uncaria guianensis (Aubl.) J. F. Gmeland on Primary Endometriotic Cells: A 2D and 3D Study.

There is increasing interest in the potential of natural compounds to treat diseases, such as endometriosis, a gynecological disorder that affects 10-15% of women of reproductive age, and it is related to severe pelvic pain and infertility. We have evaluated the in vitro effects of rutin and the aqueous bark, roots, and leaf extracts (ABE, ARE, and ALE, respectively) and isolated components of Uncaria guianensis on stromal cells from eutopic endometrium and lesions of patients with endometriosis. Two- and three-dimensional cultures were used to assess the cell death and production of reactive oxygen species (ROS), cytokines and growth factors of cells following exposure to these natural products. The applied treatments did not reduce cellular viability, but ROS production did increase. In addition, significant increases in the levels of interleukin (IL)-15, IL-17A, IL-4, IL-6, tumor necrosis factor-α, and vascular endothelium growth factor were observed when 2D-cells from endometrium of patients with endometriosis were treated with ABE, while exposure to ALE induced significant increases in epidermal growth factor in lesion cells.

A single muscle contusion promotes an immediate alteration in mitochondrial bioenergetics response in skeletal muscle fibres with different metabolism.

Skeletal muscle is the most abundant tissue in the human body and mechanical injuries are common; these are frequently of mechanical origins, such as contusion. However, the immediate mitochondrial response to injury and energetic substrate utilisation is still unclear. We evaluated the acute response in mitochondrial function after a single muscle contusion, either in fast twitch fibres (glycolytic metabolism), fast and slow twitch (oxidative and glycolytic metabolism), or slow twitch fibres (oxidative metabolism). Rats were assigned to two groups: control and Lesion (muscle contusion). We collected the gastrocnemius and soleus muscles. The fibres were analysed for mitochondrial respiration, lactate dehydrogenase (LDH), citrate synthase (CS) activity, Ca2+ uptake, and H2O2 production. We found that muscle injury was able to increase ATP synthesis-dependent and OXPHOS oxygen flux in the oxidative fibres when stimulated by complex I + II substrates. On the other hand, the muscle injury increased hydrogen peroxide (H2O2) production when compared to control fibres, and reduced citrate synthase activity; however, it did not change Ca2+ uptake. Surprisingly, injury in mixed fibres increased the OXPHOS and ATP synthesis oxygen consumption, and H2O2 production, but it reduced Ca2+ uptake. The injury in glycolytic fibres did not affect oxygen flux coupled to ATP synthesis, citrate synthase, and lactate dehydrogenase activity, but did reduce Ca2+ uptake. Finally, we demonstrated distinct mitochondrial responses between the different muscle fibres, indicating that the mitochondrial dynamics is related to flexibilities in metabolism, and that reactive oxygen species directly affect physiology and normal function.

Understanding the Metabolic Profile of Macrophages During the Regenerative Process in Zebrafish.

In contrast to mammals, lower vertebrates, including zebrafish (Danio rerio), have the ability to regenerate damaged or lost tissues, such as the caudal fin, which makes them an ideal model for tissue and organ regeneration studies. Since several diseases involve the process of transition between fibrosis and tissue regeneration, it is necessary to attain a better understanding of these processes. It is known that the cells of the immune system, especially macrophages, play essential roles in regeneration by participating in the removal of cellular debris, release of pro- and anti-inflammatory factors, remodeling of components of the extracellular matrix and alteration of oxidative patterns during proliferation and angiogenesis. Immune cells undergo phenotypical and functional alterations throughout the healing process due to growth factors and cytokines that are produced in the tissue microenvironment. However, some aspects of the molecular mechanisms through which macrophages orchestrate the formation and regeneration of the blastema remain unclear. In the present review, we outline how macrophages orchestrate the regenerative process in zebrafish and give special attention to the redox balance in the context of tail regeneration.

In vitro and computational studies showed that perezone inhibits PARP-1 and induces changes in the redox state of K562 cells.

Cancer is one of the leading causes of morbidity and mortality worldwide. This disease is characterized by uncontrolled growth and proliferation of abnormal cells with a high probability to develop metastasis. Recently, it was demonstrated that perezone, a sesquiterpene quinone, is capable to induce cell death in leukemia (K562), prostate (PC-3), colorectal (HCT-15) and lung (SKLU-1) cancer cell lines; however, its mechanism of action is unknown. Therefore, in this study, in vitro and computational studies were performed to determine the mechanism of action of perezone. Firstly, changes in K562 cell viability, as well as changes in the redox status of the cell in response to treatment with several concentrations of perezone were analyzed. The type of cell death induced, and the modification of the cell cycle were determined. In addition, MD simulations and docking studies were performed to investigate the interaction of perezone with seven regulators of the apoptotic process. Finally, the ability of perezone to inhibit PARP-1 was evaluated by in vitro studies. K562 cells treated with perezone exhibited decreased viability and more oxidized status, being this effect concentration-dependent. In addition, the increase of G0/G1 phase of cell cycle and apoptosis were observed. According to the performed computational studies conducted, perezone showed the highest affinity to PARP-1 enzyme being this complex the most stable due to the presence of a small and deep cavity in the active site, which allows perezone to fit deeply by forming hydrogen bonds and hydrophobic interactions, which drive this interaction. The activity of perezone as PARP-1 inhibitor was corroborated with an IC50 = 181.5 µM. The pro-apoptotic action of perezone may be related to PARP-1 inhibition and changes in the redox state of the cell. The obtained results allowed to understand the biological effect of perezone and, consequently, these could be employed to develop novel PARP-1 inhibitors.

Increased ROS generation causes apoptosis-like death: Mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex.

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm = bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(η2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(η2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(η2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

6-Hydroxydopamine induces different mitochondrial bioenergetics response in brain regions of rat.

Mitochondrial dysfunction has been demonstrated to have a central role in Parkinson Disease (PD) pathophysiology. Some studies have indicated that PD causes an impairment in mitochondrial bioenergetics; however, the effects of PD on brain-region specific bioenergetics was never investigated before. This study aimed to evaluate mitochondrial bioenergetics in different rat brain structures in an in vitro model of PD using 6-OHDA. Rat brain slices of hippocampus, striatum, and cortex were exposed to 6-OHDA (100 µM) for 1 h and mitochondrial bioenergetic parameters, peroxide production, lactate dehydrogenase (LDH) and citrate synthase (CS) activities were analyzed. Hippocampus slices exposed to 6-OHDA presented increased peroxide production but, no mitochondrial adaptive response against 6-OHDA damage. Cortex slices exposed to 6-OHDA presented increased oxygen flux related to oxidative phosphorylation and energetic pathways exchange demonstrated by the increase in LDH activity, suggesting a mitochondrial compensatory response. Striatum slices exposed to 6-OHDA presented a decrease of oxidative phosphorylation and decrease of oxygen flux related to ATP-synthase indicating an impairment in the respiratory chain. The co-incubation of 6-OHDA with n-acetylcysteine (NAC) abolished the effects of 6-OHDA on mitochondrial function in all brain regions tested, indicating that the increased reactive oxygen species (ROS) production is responsible for the alterations observed in mitochondrial bioenergetics. The present results indicate a brain-region specific response against 6-OHDA, providing new insights into brain mitochondrial bioenergetic function in PD. These findings may contribute to the development of future therapies with a target on energy metabolism.

Increased ROS generation causes apoptosis-like death: mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm=bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(n2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(n2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(n2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

Methylmercury and diphenyl diselenide interactions in Drosophila melanogaster: effects on development, behavior, and Hg levels.

Methylmercury (MeHg) is a highly toxic environmental pollutant which binds with a high affinity to selenol groups. In view of this, seleno-compounds have been investigated as MeHg antidotes. In the present study, we evaluated the effects of the co-exposure to MeHg and the seleno-compound diphenyl diselenide (PhSe)2 on Drosophila melanogaster. We measured the survival rate, developmental survival, locomotor ability, reactive oxygen species (ROS) production, and Hg levels in D. melanogaster exposed to MeHg and/or (PhSe)2 in the food. Exposure to MeHg caused a reduction in the survival rate, developmental survival, and locomotion in D. melanogaster. In addition, MeHg increased the ROS production and mercury levels in flies. The co-exposure to MeHg and (PhSe)2 did not prevent the toxic effects of MeHg in D. melanogaster. On the contrary, the co-exposure enhanced the toxic effects on the locomotor ability and developmental survival. This effect may be explained by the fact that the co-exposure increased the Hg levels in body when compared to flies exposed only to MeHg, suggesting that MeHg and (PhSe)2 interaction may increase Hg body burden in D. melanogaster which could contribute for the increased toxicity observed in the co-exposure.

Addition of insulin-like growth factor I to the maturation medium of bovine oocytes subjected to heat shock: effects on the production of reactive oxygen species, mitochondrial activity and oocyte competence.

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts.