Antioxidant responses driven by Hesperetin and Hesperidin counteract Parkinson's disease-like phenotypes in Drosophila melanogaster.

The study investigated the protective effects of Hesperetin (HSP) and Hesperidin (HSD) on 1 methyl, 4 phenyl, 1,2,3,6 tetrahydropyridine hydrochloride (MPTP)-induced Parkinsonism in Drosophila melanogaster (D. melanogaster). After a lifespan study to select exposure time and concentrations, flies were co-exposed to MPTP (0.4 mg/g diet), Hesperetin (0.2 and 0.4 mg/g diet), and Hesperidin (0.1 and 0.4 mg/g) for 7 days. In addition to in vivo parameters, we assayed some markers of oxidative stress and antioxidant status (lipid peroxidation, protein carbonylation, thiol content, hydrogen peroxide, and nitrate/nitrite levels, mRNA expression of Keap-1 (Kelch-like ECH associated protein 1), /Nrf2 (Nuclear factor erythroid 2 related factor 2), catalase, and glutathione-S-transferase (GST) activities), and cholinergic (acetyl cholinesterase activity (AChE) and dopaminergic signaling content and the mRNA expression of tyrosine hydroxylase (TH), monoamine oxidase (MAO-like) activity). In addition to increasing the lifespan of flies, we found that both flavonoids counteracted the adverse effects of MPTP on survival, offspring emergence, and climbing ability of flies. Both flavonoids also reduced the oxidative damage on lipids and proteins and reestablished the basal levels of pro-oxidant species and activities of antioxidant enzymes in MPTP-exposed flies. These responses were accompanied by the normalization of the mRNA expression of Keap1/Nrf2 disrupted in flies exposed to MPTP. MPTP exposure also elicited changes in mRNA expression and content of TH as well as in MAO and AChE activity, which were reversed by HST and HSD. By efficiently hindering the oxidative stress in MPTP-exposed flies, our findings support the promising role of Hesperetin and Hesperidin as adjuvant therapy to manage Parkinsonism induced by chemicals such as MPTP.

JM-20 potently prevents the onset of caffeine-induced anxiogenic phenotypes in zebrafish (Danio rerio).

Anxiety is among the most prevalent mental disorders present in the general population. Benzodiazepines are the most commonly prescribed drugs for the treatment of anxiety. Using zebrafish as a model organism, we investigated the anxiolytic activity of JM-20, a novel hybrid molecule with a 1,5-benzodiazepine ring fused to a dihydropyridine moiety. Firstly, we carried out some assays to analyze the possible toxicity mediated by JM-20. For this, zebrafish were exposed to different JM-20 concentrations (0-5 µM) for 96 h. Then, using the novel tank test, we evaluated both locomotor and anxiety-like behavior of the animals. Furthermore, brain, liver and plasma were removed to assess toxicity parameters. JM-20 exposure did not cause changes on novel tank, and also did not alter brain viability, hepatic LDH and plasma ALT levels. Afterward, we investigated whether a pre-exposure to JM-20 would prevent the anxiogenic effect evoked by caffeine. In the novel tank test, caffeine significantly decreased the time spent at the top, as well as the number of transitions to the top area. Moreover, caffeine decreased both the total and average time spent in the lit area, as well as increased the number of risk episodes evaluated by the light-dark test. Whole-body cortisol levels were also increased by caffeine exposure. Interestingly, pre-treatment with JM-20 abolished all alterations induced by caffeine. The anxiolytic effect profile of JM-20 was similar to those found for diazepam (positive control). Our findings show, for the first time, the anxiolytic effect of JM-20 in zebrafish, and its relationship with cortisol regulation.

EtHg is more toxic than MeHg to human peripheral blood mononuclear cells: Involvement of apoptotic, mitochondrial, oxidative and proliferative parameters.

BACKGROUND: Methylmercury (MeHg) and ethylmercury (EtHg) are potent toxicants affecting the environment and human healthy. In this way, the present study aimed to investigate and compare the effects of MeHg and EtHg exposure on human peripheral blood mononuclear cells (PBMCs), which are critical components of the mammalian immune system. METHODS: PBMCs were exposed to 2.5 µM MeHg or 2.5 µM EtHg. The number of cells and incubation times varied according to each assay. After exposures, the PBMCs were subjected to different evaluations, including cell viability, morphological aspects, cell cycle phases, indices of apoptosis and necrosis, reactive species (RS) production, and mitochondrial functionality. RESULTS: PBMCs exposed to EtHg were characterized by decreased viability and size, increased granularity, RS production, and apoptotic indexes accompanied by an intensification of Sub-G1 and reduction in G0-G1 cell cycle phases. Preceding these effects, we found mitochondrial dysfunctions, namely a reduction in the electron transport system related to mitochondrial complex I. In contrast, PBMCs exposed to MeHg showed only reduced viability. By ICP-MS, we found that PBMCs treated with EtHg accumulated Hg + levels ∼1.8-fold greater than MeHg-exposed cells. CONCLUSIONS AND SIGNIFICANCE: Taken together, our findings provide important insights about mercury immunotoxicity, showing that EtHg is more immunotoxic to human PBMCs than MeHg.

Kaempferol counteracts toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in D. melanogaster: An implication of its mitoprotective activity.

The current study aimed to investigate whether kaempferol (KMP), the major bioactive component of green leafy vegetables, could counteract the toxicity elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in Drosophila melanogaster or not. First, we performed a dose-response curve, where adult wild-type flies were fed on diet-containing different concentrations of KMP throughout their lifespan. Afterward, flies were fed on a diet containing MPTP (500 µM) and KMP (20 and 40 µM) for 7 days. The MPTP- fed flies presented a higher mortality rate, lower emergence rate, locomotor deficits, and disruption in circadian rhythm when compared to the control. MPTP exposure induced severe oxidative stress, which was marked by reduction in thiol content, overproduction of reactive species, lipid and protein oxidation, and disruption of enzymes of antioxidant and neurotransmission pathways. MPTP also compromised the mitochondrial dynamics and respiration of flies, affecting the electron transport chain, oxidative phosphorylation, and fusion/fission processes. Besides extending per se the lifespan of flies, KMP counteracted the toxic effects of MPTP on the circadian cycle, survival, climbing, and hatching rates. KMP was also effective in restoring the activities of acetylcholinesterase (AChE) and monoamine oxidase (MAO) enzymes, as well as in normalizing the levels of all oxidant/antioxidant markers disrupted in MPTP-fed flies. Indeed, KMP reestablished the mitochondrial functionality in MPTP- fed flies, restoring the electron transport system linked to mitochondrial complex I and II, and rescuing the mRNA transcription of genes associated with mitochondrial fusion and fission, namely OPA-1 (Optic atrophy 1) and DRP-1 (Dynamin related protein 1). Our results showed the efficacy of KMP in hindering the toxicity induced by MPTP in D. melanogaster and suggest that the mitoprotective action of flavonoid may be boosting its anti-parkinsonism activity in the model. Besides, the study showed that wild-type strains of D. melanogaster proved to be reproducible in vivo model to mimic parkinsonian phenotypes through exposure to the neurotoxin MPTP.

Environmentally relevant manganese concentrations evoke anxiety phenotypes in adult zebrafish.

Manganese (Mn) is an essential metal for living organisms. However, the excess of Mn can be toxic, especially for the central nervous system. Herein, we used adult zebrafish as model organism to investigate the relationship of an environmentally relevant Mn exposure with the onset of neurobehavioral disturbances and brain biochemical alterations. Fish were exposed to MnCl2 at 0.5, 2.0, 7.5 and 15.0 mg/L for 96 h, and after submitted to trials for examining exploratory, locomotor and anxiety-related behaviors. The neurobehavioral parameters were followed by the analyses of cell viability, Mn accumulation and acetylcholinesterase activity in the brain, and whole-body cortisol levels. By Novel tank, Light dark and Social preference test, we found that the exposure to Mn, along with locomotor deficits induced anxiety-like phenotypes in zebrafish. Most of these behavioral changes were evoked by the highest concentrations, which also caused cell viability loss, higher accumulation of Mn and increased AChE activity in the brain, and an increase in the whole-body cortisol content. Our findings demonstrated that zebrafish are quite sensitive to levels of Mn found in the environment, and that the magnitude of the neurotoxic effects may be associated with the levels of manganese accumulated in the brain. Interestingly, we showed that Mn exposure in addition to motor deficits may also cause psychiatric abnormalities, namely anxiety.

Methylglyoxal disrupts the functionality of rat liver mitochondria.

Methylglyoxal (MG) is a reactive metabolite derived from different physiological pathways. Its production can be harmful to cells via glycation reactions of lipids, DNA, and proteins. But, the effects of MG on mitochondrial functioning and bioenergetic responses are still elusive. Then, the effects of MG on key parameters of mitochondrial functionality were examined here. Isolated rat liver mitochondria were exposed to 0.1-10 mM of MG to determine its toxicity in the mitochondrial viability, membrane potential (Δψm), swelling and the superoxide (O2•-) production. Besides, mitochondrial oxidative phosphorylation parameters were analyzed by high-resolution respiratory (HRR) assay. In this set of experiments, routine state, PM state (pyruvate/malate), oxidative phosphorylation (OXPHOS), LEAK respiration, electron transport system (ETS) and oxygen residual (ROX) states were evaluated. HRR showed that PM state, OXPHOS CI-Linked, LEAK respiration, ETS CI/CII-Linked and ETS CII-Linked/ROX were significantly inhibited by MG exposure. MG also inhibited the complex II activity, and decreased Δψm and the viability of mitochondria. Taken together, our data indicates that MG is an inductor of mitochondrial dysfunctions and impairs important steps of respiratory chain, effects that can alter bioenergetics responses.

Syzygium cumini leaf extract protects macrophages against the oxidized LDL-induced toxicity: A promising atheroprotective effect.

Oxidized LDL (oxLDL) plays a pivotal role on atherosclerosis development, mainly in the formation of lipid-laden macrophage "foam cells". As a consequence, substances that can modulate LDL oxidation have a pharmacological and therapeutic relevance. Based in previous findings showing the ability of Syzigium cumini leaf extract (ScExt) in preventing LDL oxidation in vitro, this study was aimed to assess the effects of ScExt on oxLDL-mediated toxicity in murine J774 macrophages-like cells. For biochemical analyses, LDL isolated from fresh human plasma and oxidized with CuSO4 was incubated with ScExt pre-treated macrophages. Our results demonstrated that ScExt was efficient in preventing the overproduction of reactive oxygen/nitrogen species (ROS/RNS), the loss of macrophage's viability and the foam cells formation induced by oxLDL. These protective effects of ScExt make it a promising antioxidant for future trials toward atherogenesis.

Methylglyoxal disturbs DNA repair and glyoxalase I system in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a highly reactive aldehyde able to form covalent adducts with proteins and nucleic acids, disrupting cellular functions. In this study, we performed a screening of Saccharomyces cerevisiae (S. cerevisiae) strains to find out which genes of cells are responsive to MG, emphasizing genes against oxidative stress and DNA repair. Yeast strains were grown in the YPD-Galactose medium containing MG (0.5 to 12 mM). The tolerance to MG was evaluated by determining cellular growth and cell viability. The toxicity of MG was more pronounced in the strains with deletion in genes engaged with DNA repair checkpoint proteins, namely Rad23 and Rad50. MG also impaired the growth and viability of S. cerevisiae mutant strains Glo1 and Gsh1, both components of the glyoxalase I system. Differently, the strains with deletion in genes encoding for antioxidant enzymes were apparently resistant to MG. In summary, our data indicate that DNA repair and MG detoxification pathways are keys in the control of MG toxicity in S. cerevisiae.

Methylglyoxal disturbs the expression of antioxidant, apoptotic and glycation responsive genes and triggers programmed cell death in human leukocytes.

Methylglyoxal (MG) is a α-dycarbonyl compound derived mainly from glycolysis, whose accumulation is harmful for cells and tissues. Here, we evaluated the cytotoxic effects induced by MG in leukocytes after an acute exposure, measuring as endpoints of toxicity some markers of oxidative stress and programmed cell death. Human leukocytes were isolated and incubated with MG at concentrations ranging from 0.1 to 10 mM for 2.5 h, and subsequently prepared for assays based in flow cytometry, gene expression and immunoreactivity profile. The cells exposed to higher concentrations of MG had significant loss of viability, increased reactive species (RS) production and apoptosis/necrosis rate. These phenomena were accompanied by morphological changes (increased size and granularity) and disruption in mRNA expression of antioxidant, apoptotic and glycation-responsive genes, particularly: Nrf2 (Nuclear factor (erythroid-derived 2)-like 2), SOD1 (CuZn-superoxide dismutase), SOD2 (Mn-superoxide dismutase), GSR (glutathione-S-reductase), BAX (BAX-associated X protein), BCL-2 (BCL-2-associated X protein), AIF (apoptosis inducing factor), GLO-1 (glyoxalase-1) and RAGE (receptor for advanced glycation end products). The mRNA expression of CASP 9 and CASP 3 (caspase-9 and 3) as well as the immunoreactivity of proteins were not changed by MG. Collectively, our data provide evidence that MG activates programmed cell death pathways in leukocytes and that this effect seems to be associated with disturbances in cell redox signaling.

Safety profile of AZT derivatives: Organoselenium moieties confer different cytotoxic responses in fresh human erythrocytes during in vitro exposures.

INTRODUCTION: The incorporation of selenium in the structure of nucleosides is a promising strategy to develop novel therapeutic molecules. OBJECTIVE: To assess the toxic effects of three AZT derivatives containing organoselenium moieties on human erythrocytes. METHODOLOGY: Freshly human erythrocytes were acutely treated with AZT and selenium derivatives SZ1 (chlorophenylseleno), SZ2 (phenylseleno) and SZ3 (methylphenylseleno) at concentrations ranging from 10 to 500 µM. Afterwards, parameters related to membrane damage, redox dyshomeostasis and eryptosis were determined in the cells. RESULTS: The effects of AZT and derivatives toward erythrocytes differed considerably. Overall, the SZ3 exhibited similar effect profiles to the prototypal AZT, without causing cytotoxicity. Contrary, the derivative SZ1 induced hemolysis and increased the membrane fragility of cells. Reactive species generation, lipid peroxidation and thiol depletion were also substantially increased in cells after exposure to SZ1. δ-ALA-D and Na+/K+-ATPase activities were inhibited by derivatives SZ1 and SZ2. Additionally, both derivatives caused eryptosis, promoting cell shrinkage and translocation of phosphatidylserine at the membrane surface. The size and granularity of erythrocytes were not modified by any compound. CONCLUSION: The insertion of either chlorophenylseleno or, in a certain way, phenylseleno moietes in the structure of AZT molecule was harmful to erythrocytes and this effect seems to involve a pro-oxidant activity. This was not true for the derivative encompassing methylphenylseleno portion, making it a promising candidate for pharmacological studies.

Evaluation of methylglyoxal toxicity in human erythrocytes, leukocytes and platelets.

Methylglyoxal (MG) is a reactive dicarbonyl metabolite originated mainly from glucose degradation pathway that plays an important role in the pathogenesis of diabetes mellitus (DM). Reactions of MG with biological macromolecules (proteins, DNA and lipids) can induce cytotoxicity and apoptosis. Here, human erythrocytes, leukocytes and platelets were acutely exposed to MG at concentration ranging from 0.025 to 10 mM. Afterwards, hemolysis and osmotic fragility in erythrocytes, DNA damage and cell viability in leukocytes, and the activity of purinergic ecto-nucleotidases in platelets were evaluated. The levels of glycated products from leukocytes and free amino groups from erythrocytes and platelets were also measured. MG caused fragility of membrane, hemolysis and depletion of amino groups in erythrocytes. DNA damage, loss of cell viability and increased levels of glycated products were observed in leukocytes. In platelets, MG inhibited the activity of enzymes NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) without affecting the levels of free amino groups. Our findings provide insights for understanding the mechanisms involved in MG acute toxicity towards distinct blood cells.

Diphenyl ditelluride intoxication triggers histological changes in liver, kidney, and lung of mice.

Tellurium compounds may be cytotoxic to different cells types. Thus, this work evaluated the effect of diphenyl ditelluride ((PhTe)2), an organotellurium commonly used in organic synthesis, on the morphology of liver, kidney, and lung. Adult mice were acutely (a subcutaneous single dose: 250 µmol/kg) or subchronically (one daily subcutaneous dose: 10 or 50 µmol/kg for 7 and 14 days) exposed to (PhTe)2. Afterwards, the histological analyses of liver, kidney, and lungs were performed. Liver histology revealed that the hepatocytes of mice subchronically exposed to (PhTe)2 presented cytoplasmic vacuolization, hydropic degeneration, and hyperchromatic nuclei. Subchronic exposure to 50 µmol/kg (PhTe)2 also caused hepatic necrosis. Microvesicular and macrovesicular steatosis were identified in liver of mice acutely exposed to (PhTe)2. Acute and subchronic intoxication with (PhTe)2 induced changes on epithelial cells of renal tubules, namely, loss of brush border and cytoplasmatic vacuolization. Atrophy and hypertrophy, cast proteinaceous formation, and acute tubular necrosis were also identified in renal tissue. Mice subchronically exposed to 50 µmol/kg (PhTe)2 developed intra-alveolar edema and alveolar wall congestion in some areas of lungs. Acute exposure to (PhTe)2 did not cause histological changes in lungs. Our data show that (PhTe)2 may be considered a histotoxic agent for liver, kidney, and lung.