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Background: COVID-19, caused by SARS-CoV-2, is a potentially lethal, rapidly-expanding pandemic and many efforts are being carried out worldwide to understand and control the disease. COVID-19 patients may display a cytokine release syndrome, which causes severe lung inflammation, leading, in many instances, to death. Objective: This paper is intended to explore the possibilities of controlling the COVID-19-associated hyperinflammation by using licensed drugs with anti-inflammatory effects. Hypothesis: We have previously described that pentoxifylline alone, or in combination with oxypurinol, reduces the systemic inflammation caused by experimentally-induced pancreatitis in rats. Pentoxifylline is an inhibitor of TNF-α production and oxypurinol inhibits xanthine oxidase. TNF-α, in turn, activates other inflammatory genes such as Nos2, Icam or IL-6, which regulate migration and infiltration of neutrophils into the pulmonary interstitial tissue, causing injury to the lung parenchyma. In acute pancreatitis, the anti-inflammatory action of pentoxifylline seems to be mediated by the prevention of the rapid and presumably transient loss of PP2A activity. This may also occur in the hyperinflammatory -cytokine releasing phase- of SARS-CoV-2 infection. Therefore, it may be hypothesized that early treatment of COVID-19 patients with pentoxifylline, alone or in combination with oxypurinol, would prevent the potentially lethal acute respiratory distress syndrome. Conclusion: Pentoxifylline and oxypurinol are licensed drugs used for diseases other than COVID-19 and, therefore, phase I clinical trials would not be necessary for the administration to SARS-CoV-2- infected people. It would be worth investigating their potential effects against the hyperinflammatory response to SARS-CoV-2 infection.

Permethrin (PM) is one of the most used synthetic pyrethroid worldwide. Exposure to this compound during pregnancy and early childhood has been indicated as a risk factor for neurodevelopmental disorders. We evaluated the long-term effects of embryonic PM exposure in different stages of zebrafish development. Briefly, embryos (3 hpf) were exposed to sub-lethal concentrations of PM (25 and 50 μg.L−1) during 24 h and then behavioral parameters were evaluated during embryonic (28 hpf), eleutheroembryonic (3 dpf), larval (7 dpf), and adult stages (90 dpf). PM exposure decreased spontaneous movement at 28 hpf and decreased thigmotaxis in eleutheroembryos. The long-term effects of PM include changes in non-motor behaviors such as fear and anxiety in larva and adults. Adults embryonically exposed to PM also showed a significant increase in aggressiveness parameters. These results demonstrated that embryonic exposure to PM induces persistent neurotoxic effects in adulthood, which can impair the cognitive and behavioral fitness of non-target species contributing to a rise in neurodevelopmental disorders.

Chlorpyrifos (CP) is an organophosphate insecticide widely used in the control of agriculture and domestic pests. Occupational exposure is a major form of human poisoning by organophosphates and current therapies for these compounds are not completely efficient. Psidium guajava is a plant widely used in folk medicine and its antioxidant activity has been described. In this study we evaluated the antioxidant and protective potential of the hydroalcoholic extract of P. guajava (HEPG) against CP induced toxicity in the fruit fly Drosophila melanogaster. HEPG in vitro antioxidant activity was confirmed by ABTS, DPPH, Total Phenolics and FRAP assays. The exposure of flies to CP caused increased mortality, locomotor deficits and inhibition of acetylcholinesterase. Flies exposed to CP presented elevated ROS and lipid peroxidation which was accompanied by a significant decrease in mitochondrial viability. As a response to increased oxidative stress, CP exposed flies showed increased GST activity and GSH levels. The mRNA expression of NRF2 and MPK2 (which encodes D. melanogaster p38MAPK) was also significantly up-regulated. HEPG was able to restore all the damage and biochemical/molecular alterations caused by CP. Our results show for the first time the P. guajava potential protective effect against the toxicity caused by chlorpyrifos. Keywords: Organophosphate compound, Oxidative stress, Natural compounds, Protective effects

Mitochondria play an important role in cell life and in the regulation of cell death. In addition, mitochondrial dysfunction contributes to a wide range of neuropathologies. The nucleoside Guanosine (GUO) is an endogenous molecule, presenting antioxidant properties, possibly due to its direct scavenging ability and/or from its capacity to activate the antioxidant defense system. GUO demonstrate a neuroprotective effect due to the modulation of the glutamatergic system and maintenance of the redox system. Thus, considering the few studies focused on the direct effects of GUO on mitochondrial bioenergetics, we designed a study to evaluate the in vitro effects of GUO on rat mitochondrial function, as well as against Ca2+-induced impairment. Our results indicate that GUO prevented mitochondrial dysfunction induced by Ca2+ misbalance, once GUO was able to reduce mitochondrial swelling in the presence of Ca2+, as well as ROS production and hydrogen peroxide levels, and to increase manganese superoxide dismutase activity, oxidative phosphorylation and tricarboxylic acid cycle activities. Our study indicates for the first time that GUO could direct prevent the mitochondrial damage induced by Ca2+ and that these effects were not related to its scavenging properties. Our data indicates that GUO could be included as a new pharmacological strategy for diseases linked to mitochondrial dysfunction.

Hepatic encephalopathy is a pathophysiological complication of acute liver failure, which may be triggered by hepatotoxic drugs such as acetaminophen (APAP). Although APAP is safe in therapeutic concentration, APAP overdose may induce neurotoxicity, which is mainly associated with oxidative stress. Caffeine is a compound widely found in numerous natural beverages. However, the neuroprotective effect of caffeine remains unclear during APAP intoxication. The present study aimed to investigate the possible modulatory effects of caffeine on brain after APAP intoxication. Mice received intraperitoneal injections of APAP (250 mg/kg) and/or caffeine (20 mg/kg) and, 4 h after APAP administration, samples of brain and blood were collected for the biochemical analysis. APAP enhanced the transaminase activity levels in plasma, increased oxidative stress biomarkers (lipid peroxidation and reactive oxygen species), promoted an imbalance in endogenous antioxidant system in brain homogenate and increased the mortality. In contrast, APAP did not induce dysfunction of the mitochondrial bioenergetics. Co-treatment with caffeine modulated the biomarkers of oxidative stress as well as antioxidant system in brain. Besides, survival assays demonstrated that caffeine protective effects could be dose- and time-dependent. In addition, caffeine promoted an increase of mitochondrial bioenergetics response in brain by the enhancement of the oxidative phosphorylation, which could promote a better energy supply necessary for brain recovery. In conclusion, caffeine prevented APAP-induced biochemical alterations in brain and reduced lethality in APAP-intoxicated mice, these effects may relate to the preservation of the cellular antioxidant status, and these therapeutic properties could be useful in the treatment of hepatic encephalopathy induced by APAP intoxication.

Parkinson’s disease is a degenerative and progressive illness characterized by the degeneration of dopaminergic neurons. 6-hydroxydopamine (6-OHDA) is a widespread model for induction of molecular and behavioral alterations similar to Parkinson and has contributed for testing of compounds with neuroprotective potential. The Brazilian plant Anacardium microcarpum is used in folk medicine for treatment of several illnesses; however, the knowledge about toxicology and biological effects for this plant is very rare. The neuroprotective effect from hydroalcoholic extract and methanolic and acetate fraction of A. microcarpum on 6-OHDA-induced damage on chicken brain slices was investigated in this study. 6-OHDA decreased cellular viability measured by MTT reduction assay, induced lipid peroxidation by HPLC, stimulated Glutathione-S-Transferase and Thioredoxin Reductase activity, and decreased Glutathione Peroxidase activity and the total content of thiols containing compounds. The methanolic fraction of A. microcarpum presented the better neuroprotective effects in 6-OHDA-induced damage in relation with hydroalcoholic and acetate fraction. The presence of AKT and ERK1/2 pharmacological inhibitors blocked the protective effect of methanolic fraction suggesting the involvement of survival pathways in the neuroprotection by the plant. The plant did not prevent 6-OHDA autoxidation or 6-OHDA-induced mitochondrial dysfunction. Thus, the neuroprotective effect of the methanolic fraction of A. microcarpum appears to be attributed in part to chelating properties of extract toward reactive species and is dependent on ERK1/2 and AKT phosphorylation. This study contributes to the understanding of biochemical mechanisms implied in neuroprotective effects of the vegetal species A. microcarpum.

We conducted an investigation to evaluate the effects of Brazilian Pampa biome honey and its major phenolic compounds on the development of an erected wings posture phenotype and related mitochondrial aspects induced by Hypoxia/Reoxygenation (H/R) in Drosophila melanogaster. Flies were pre-treated for 3 days with a 10% honey solution and different concentrations of caffeic acid and ρ-coumaric acid and then submitted to hypoxia for 3 h. We observed that after reoxygenation, some flies acquired an erected wings posture and that this feature may be related to mortality. In addition, H/R induced down-regulation of ewg mRNA expression, which could be associated to the observed complex phenotype. H/R also caused a dysregulation in opa1-like, ldh and diap genes expression and reduced O2 fluxes in flie's mitochondria. Honey mitigated opa1-like mRNA expression changes provoked by H/R. Differently from honey, caffeic and ρ-coumaric acids displayed no protective effects. In conclusion, we report for the first time the protective effects of honey against complex phenotypes and mitochondrial changes induced by H/R in adult flies.

Mancozeb (MZ), a manganese/zinc-containing ethylene-bis-dithiocarbamate (EBCD) fungicide has been claimed to present low acute toxicity and short environmental persistence, however, its effects on embryogenesis in non-target organisms is unclear. Here, we used zebrafish embryos (5 hpf) to assess the potential embryotoxic effects induced by MZ (up to 72 hpf) as well as the role of reactive oxygen species (ROS) in this process by pre-treatment with a classical antioxidant (N-acetylcysteine, NAC). Markers of reactive oxygen species production (ROS), glutathione (GSH) levels and glutathione S-transferase (GST) activity were measured along with genotoxicity (comet assay), cell death (Acridine Orange) and behavioral parameters (spontaneous movement, touch stimulation and swimming response), in order to determine potential mechanisms of embryotoxicity. According to results, MZ was able to induce morphological abnormalities such as body axis distortion, DNA damage, cell death, increased ROS generation and changes in behavioral endpoints during zebrafish development. All these toxic effects were inhibited by the pre-treatment with NAC indicating a key role of redox unbalance during MZ-induced embryotoxicity. At least in our knowledge, this is the first report on the deleterious effect of MZ to the normal embryogenesis of zebrafish. In addition, the importance of ROS generation during this pathophysiological condition was highlighted.

Many studies have shown the effects of sleep deprivation in several aspects of health and disease. However, little is known about how mitochondrial bioenergetics function is affected under this condition. To clarify this, we developed a simple model of short-term sleep deprivation, in which fruit-flies were submitted to a nocturnal light condition and then mitochondrial parameters were assessed by high resolution respirometry (HRR). Exposure of flies to constant light was able to alter sleep patterns, causing locomotor deficits, increasing ROS production and lipid peroxidation, affecting mitochondrial activity, antioxidant defense enzymes and caspase activity. HRR analysis showed that sleep deprivation affected mitochondrial bioenergetics capacity, decreasing respiration at oxidative phosphorylation (OXPHOS) and electron transport system (ETS). In addition, the expression of genes involved in the response to oxidative stress and apoptosis were increased. Thus, our results suggest a connection between sleep deprivation and oxidative stress, pointing to mitochondria as a possible target of this relationship.

Mercury (Hg) is widely distributed in the environment and is known to produce several adverse effects in organisms. The aim of the present study was to examine the in vitro antioxidant activity and Hg chelating ability of the hydroalcoholic extract of Psidium guajava leaves (HEPG). In addition, the potential protective effects of HEPG against Hg(II) were evaluated using a yeast model (Saccharomyces cerevisiae). HEPG was found to exert significant antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl scavenger and inhibition of lipid peroxidation induced by Fe(II) assays in a concentration-dependent manner. The extract also exhibited significant Hg(II) chelating activity. In yeast, Hg(II) induced a significant decrease in cell viability. In contrast, HEPG partially prevented the fall in cell viability induced by Hg(II). In conclusion, HEPG exhibited protective effects against Hg(II)-mediated toxicity, which may be related to both antioxidant and Hg(II)-chelating activities.

Bioactive phytocompounds are studied by several bioactivities demonstrated, as their cytotoxic effects. The aim of this work was to evaluate the phytochemical profile, the toxic effect using the Drosophila melanogaster animal model and the anti-inflammatory and antimicrobial effect of the Alternanthera brasiliana (EEAB) ethanol extract. The phytochemical profile was performed using HPLC. The cytotoxic effect was evaluated in vivo using D. melanogaster. The anti-inflammatory effect was determined by neurogenic and antiedematogenic assays, and the antimicrobial activity was assayed using a microdilution method to determine the minimum inhibitory concentration (MIC) of the EEAB alone and in association with antibiotics. The main compound identified on the EEAB was luteolin (1.93%). Its cytotoxic effect was demonstrated after 24 h in the concentrations of 10, 20 and 40 mg/mL. The extract demonstrated an antiedematogenic effect, with a reduction of the edema between 35.57 and 64.17%. The MIC of the extract was ≥1.024 μg/mL, thus being considered clinically irrelevant. However, when the EEAB was associated with gentamicin, a synergism against all bacterial strains assayed was observed: Staphylococcus aureus (SA10), Escherichia coli (EC06) and Pseudomonas aeruginosa (PA24). Due to these results, the EEAB demonstrated a low toxicity in vivo and anti-inflammatory and synergistic activities. These are promising results, mainly against microbial pathogens, and the compounds identified can be a source of carbon backbones for the discovery and creation of new drugs.

Traumatic brain injury (TBI) is a highly complex multi-factorial disorder. Experimental trauma involves primary and secondary injury cascades that underlie delayed neuronal dysfunction and death. Mitochondrial dysfunction and glutamatergic excitotoxicity are the hallmark mechanisms of damage. Accordingly, a successful pharmacological intervention requires a multi-faceted approach. Guanosine (GUO) is known for its neuromodulator effects in various models of brain pathology, specifically those that involve the glutamatergic system. The aim of the study was to investigate the GUO effects against mitochondrial damage in hippocampus and cortex of rats subjected to TBI, as well as the relationship of this effect with the glutamatergic system. Adult male Wistar rats were subjected to a unilateral moderate fluid percussion brain injury (FPI) and treated 15 min later with GUO (7.5 mg/kg) or vehicle (saline 0.9%). Analyses were performed in hippocampus and cortex 3 h post-trauma and revealed significant mitochondrial dysfunction, characterized by a disrupted membrane potential, unbalanced redox system, decreased mitochondrial viability, and complex I inhibition. Further, disruption of Ca

Croton campestris A. St-Hill popularly known as “velame do campo” is a native species of the savannah from northeastern Brazil, being used in folk medicine due to its beneficial effects in the treatment of many diseases, inflammation, detoxification, gastritis, and syphilis; however, its potential use as an antidote against organophosphorus compound poisoning has not yet been shown. Here, the protective effect of the methanolic fraction of C. campestris A. St.-Hill (MFCC) in Drosophila melanogaster exposed to chlorpyrifos (CP) was investigated. Flies were exposed to CP and MFCC during 48 h through the diet. Following the treatments, parameters such as mortality, locomotor behavior, and oxidative stress markers were evaluated. Exposure of flies to CP induced significant impairments in survival and locomotor performance. In parallel, increased reactive oxygen species and lipoperoxidation occurred. In addition, the activity of acetylcholinesterase was inhibited by CP, and superoxide dismutase and glutathione S-transferase activity was induced. Treatment with MFCC resulted in a blockage of all CP-induced effects, with the exception of glutathione S-transferase. Among the major compounds found in MFCC, only gallic acid (GA) showed a protective role against CP while quercetin and caffeic acid alone were ineffective. When in combination, these compounds avoided the toxicity of CP at the same level as GA. As far as we know, this is the first study reporting the protective effect of MFCC against organophosphate toxicity in vivo and highlights the biotechnological potential of this fraction attributing a major role in mediating the observed effects to GA. Therefore, MFCC may be considered a promising source for the development of new therapeutic agents for the treatment of organophosphate intoxications.

© 2019 Published by Elsevier B.V., Acute pancreatitis is an inflammatory process of the pancreatic gland that may lead to dysregulation of the trans-sulfuration pathway. The aims of this work were firstly to study the methionine cycle as well as the trans-sulfuration pathway using metabolomic and proteomic approaches identifying the causes of this dysregulation in an experimental model of acute pancreatitis; and secondly to reveal the effects of S-adenosylmethionine administration on these pathways. Acute pancreatitis was induced by cerulein in mice, and a group of animals received S-adenosylmethionine treatment. Cerulein-induced acute pancreatitis rapidly caused marked depletion of methionine, S-adenosylmethionine, 5′-methylthioadenosine, cystathionine, cysteine, and glutathione levels in pancreas, but S-adenosylhomocysteine and homocysteine remained unchanged. Protein steady-state levels of S-adenosylhomocysteine-hydrolase and cystathionine gamma-lyase diminished but methylthioadenosine phosphorylase levels increased in pancreas with acute pancreatitis. Although cystathionine β-synthase protein levels did not change with acute pancreatitis, Nos2 mRNA and protein levels were markedly up-regulated and caused tyrosine nitration of cystathionine β-synthase in pancreas. S-adenosylmethionine administration enhanced Nos2 mRNA expression and cystathionine β-synthase nitration and triggered homocysteine accumulation in acute pancreatitis. Furthermore, S-adenosylmethionine administration promoted enrichment of the euchromatin marker H3K4me3 in the promoters of Tnf-α, Il-6, and Nos2 and enhanced the mRNA up-regulation of these genes. Accordingly, S-adenosylmethionine administration increased inflammatory infiltrate and edema in pancreas with acute pancreatitis. In conclusion, tyrosine-nitration of cystathionine β-synthase blockades the trans-sulfuration pathway in acute pancreatitis promoting homocysteine accumulation upon S-adenosylmethionine treatment., This work was supported by Grant SAF2015-71208-R with FEDER funds from the Spanish Ministry of Economy and Competitiveness to J.S.

Eugenia uniflora is used in the Brazilian folk medicine to treat intestinal disorders and hypertension. However, scanty information exist on its potential toxicity to human, and little is known on its antioxidant activity in biological system. Hence, we investigated for the first time the potential toxic effects of ethanolic extract (EtOH) of E. uniflora (EEEU) in human leukocytes and erythrocytes, as well as its influence on membrane erythrocytes osmotic fragility. In addition, EEEU was chemically characterized and its antioxidant capacity was evaluated. We found that EEEU (1-480μg/mL) caused neither cytotoxicity nor DNA damage evaluated by Trypan blue and Comet assay, respectively. EEEU (1-480μg/mL) did not have any effect on membrane erythrocytes fragility. In addition, EEEU inhibited Fe2+-induced lipid peroxidation in rat brain and liver homogenates, and scavenged the DPPH radical. EEEU presented some polyphenolic compounds with high content such as quercetin, quercitrin, isoquercitrin, luteolin and ellagic acid, which may be at least in part responsible for its beneficial effects. Our results suggest that consumption of EEEU at relatively higher concentrations may not result in toxicity. However, further in vitro and in vivo studies should be conducted to ascertain its safety.

As agriculture expands to provide food and wellbeing to the world’s growing population, there is a simultaneous increasing concern about the use of agrochemicals, which can harm non-target organisms, mainly in the aquatic environment. The fungicide Mancozeb (MZ) has been used on a large-scale and is a potent inducer of oxidative stress. Therefore, there is an urgent need for the development of more sensitive biomarkers designed to earlier biomonitoring of this compound. Here we tested the hypothesis that behavioral changes induced by sublethal MZ concentrations would occur first as compared to biochemical oxidative stress markers. Embryos at 4 h post-fertilization (hpf) were exposed to Mancozeb at 5, 10 and 20 μg/L. Controls were kept in embryo water only. Behavioral and biochemical parameters were evaluated at 24, 28, 72, and 168 hpf after MZ exposure. The results showed that MZ significantly altered spontaneous movement, escape responses, swimming capacity, and exploratory behavior at all exposure times. However, changes in ROS steady-stead levels and the activity of antioxidant enzymes were observable only at 72 and 168 hpf. In conclusion, behavioral changes occurred earlier than biochemical alterations in zebrafish embryos exposed to MZ, highlighting the potential of behavioral biomarkers as sensitive tools for biomonitoring programs.

Mancozeb (MZ) is a broad-spectrum fungicide used worldwide in several crops. Neurological disorders in humans and animals have been associated with exposure to this compound by mechanisms still not fully understood. Drosophila melanogaster represents a reliable model in toxicological studies, presenting genetic and biochemical similarities with mammals. In this study, D. melanogaster flies were exposed for 15 days to MZ through the food (5 and 10 mg/mL). After that period, the efficiency of mitochondrial respiration complexes and metabolic markers were analyzed and evaluated. Flies presented weight loss, lower glucose, trehalose, and glycogen levels, and augmented levels of triglycerides concerning control (non-treated group). Acetyl-CoA Synthetase (ACeCS-1) and Acyl-Coenzyme Synthetase (ACSL1) contents were unchanged by MZ treatment. Mitochondrial respiration of flies was targeted by MZ treatment, evidenced by a decrease in oxygen consumption and bioenergetics rate and inhibition in mitochondrial complexes I/II. These results suppose that an impairment in mitochondrial respiration jointly with reduced levels of energetic substrates might be a mechanism involved in MZ deleterious effects, possibly by the limitation of ATP's availability, necessary for essential cellular processes.

Permethrin (PM) is a synthetic pyrethroid insecticide widely used as domestic repellent. Damage effects to nontarget organisms have been reported, particularly in the early stages of development. Studies indicate redox unbalance as secondary PM effect. Therefore, our goal was to investigate the acute PM effects on larval zebrafish. Larvae (6 days postfertilization) were exposed to PM (25–600μg/L) during 24 hours, and 50% lethal concentration was estimated. For subsequent assays, the sublethal PM concentrations of 25 and 50μg/L were used. PM increased anxiety-like behaviors according to the Novel Tank and Light-Dark tests. At the molecular level, PM induced increased ROS, which may be related to the increased lipid peroxidation, DNA damage, and apoptosis detected in PM-exposed organisms. In parallel, upregulation of the antioxidant system was detected after PM exposure, with increased superoxide dismutase, glutathione S-transferase and glutathione reductase activities, and thiol levels. The increased of Nrf2 target genes and the activation of an electrophile response element-driven reporter Tg(EPRE:LUC-EGFP) suggest that the Nrf2 pathway can mediate a fast response to PM, leading to antioxidant amplification. By using high-resolution respirometry, we found that exposure to PM decreased the oxygen consumption in all respiratory stages, disrupting the oxidative phosphorylation and inhibiting the electron transfer system, leading to decrease in bioenergetics capacity. In addition, PM led to increases of residual oxygen consumption and changes in substrate control ratio. Glucose metabolism seems to be affected by PM, with increased lactate dehydrogenase and decreased citrate synthase activities. Taken together, our results demonstrated the adverse effects of acute sublethal PM concentrations during larval development in zebrafish, causing apparent mitochondrial dysfunction, indicating a potential mechanism to redox unbalance and oxidative stress, which may be linked to the detected cell death and alterations in normal behavior patterns caused by acute PM exposure.

Organic selenium compounds have several pharmacological activities already described, as anti-inflammatory and antitumor activities, which have been attributed to their antioxidant effects. Because they are promising in pharmacology, the synthesis of these compounds has increased significantly. As many new molecules are synthesized the use of a simple model like Caenorhabditis elegans is highly advantageous for initial evaluation of the toxicity and therapeutic potential of these molecules. The objective of this study was to evaluate the toxicity and antioxidant capacity of a series of selenotriazoles compounds in C. elegans. The animals were exposed to the compounds in liquid medium for only 30 min at the first larval stage (L1). The compounds had no toxic effects at the concentrations tested. Treatment with selenotriazoles (10 μM) partially reversed the stress induced by the pesticide paraquat (1 mM). Se-Tz Ia compound partially increased the survival of worms treated with H2O2 (0.5 mM). The compounds also increased the longevity of mev-1 mutants, which have a reduced life span by the production of excessive reactive oxygen species (ROS) in the mitochondria caused by a mutation in complex II of the electron transport chain. In addition, the compounds reduced the levels of ROS determined by the fluorescent probe DCF-DA as well as also reduced catalase enzyme activity in these animals. Based on the results found, it is possible to conclude that the compounds have antioxidant activity mainly in oxidative stress condition generated by a mitochondrial dysfunction in C. elegans.

This study was designed to evaluate the effects ofBauhinia forficataLink subsp.pruinosa(BF) tea on oxidative stress and liver damage in streptozotocin (STZ)-induced diabetic mice. Diabetic male mice have remained 30 days without any treatment. BF treatment started on day 31 and continued for 21 days as a drinking-water substitute. We evaluated (1) BF chemical composition; (2) glucose levels; (3) liver/body weight ratio and liver transaminases; (4) reactive oxygen species (ROS), lipid peroxidation, and protein carbonylation in liver; (5) superoxide dismutase (SOD) and catalase (CAT) activities in liver; (6)δ-aminolevulinate dehydratase (δ-ALA-D) and nonprotein thiols (NPSH) in liver; (7) Nrf2, NQO-1, and HSP70 levels in liver and pancreas. Phytochemical analyses identified four phenols compounds. Diabetic mice present high levels of NQO-1 in pancreas, increased levels of ROS and lipid peroxidation in liver, and decrease in CAT activity. BF treatment normalized all these parameters. BF did not normalize hyperglycemia, liver/body weight ratio, aspartate aminotransferase, protein carbonyl, NPSH levels, andδ-ALA-D activity. The raised oxidative stress seems to be a potential mechanism involved in liver damage in hyperglycemic conditions. Our results indicated that BF protective effect could be attributed to its antioxidant capacity, more than a hypoglycemic potential.

We aimed to investigate the potential beneficial effects of the Brazilian Pampa biome honey in a Drosophila-based hypoxia model. Adult flies were reared in standard medium in the presence or absence of honey (at a final concentration of 10 % in medium). Then, control flies (4 % sucrose in medium) and honey-treated flies were submitted to hypoxia. Subsequently, flies were analyzed for mortality, neurolocomotor behavior (negative geotaxis), mitochondrial/oxidative stress parameters and expression of hypoxia/stress related genes by RT-qPCR. The HPLC analysis revealed the presence of phenolics and flavonoids in the studied honey. Caffeic acid was the major compound followed by p-coumaric acid and kaempferol. The presence of such compounds was correlated with a substantial antioxidant activity in vitro. Flies subjected to hypoxia presented marked mortality, locomotor deficits and changes in oxidative stress and mitochondrial activity parameters. Honey treatment was able to completely block mortality and locomotor phenotypes. In addition, honey was able to reverse ROS production and hypoxia-induced changes in mitochondrial complex I and II activity. Hypoxia also induced an up-regulation in mRNA expression of Sima (HIF-1), NFκβ, NRF2, HOX, AKT-1, InR, dILP2, dILP5 and HSP27. Honey treatment was not able to modulate changes in the tested genes, indicating that its protective effects involve additional mechanisms other than transcriptional activity of hypoxia-driven adaptive responses in flies. Our results demonstrated, for the first time, the beneficial effects of honey against the deleterious effects of hypoxia/reperfusion processes in a complex organism.

Mancozeb (MZ), a manganese- and zinc-containing ethylene-bis-dithiocarbamate, is a broad-spectrum fungicide. Harmful effects of this fungicide have been reported in nontarget organisms via a not fully understood mechanism. Drosophila melanogaster has provided remarkable contributions for toxicological studies. This work was aimed at evaluating the biochemical targets and implication of oxidative stress in MZ-mediated toxicity in drosophilas. Exposure of flies for fifteen days to MZ at 5 and 10 mg/mL through the diet impaired locomotor performance and induced fly mortality. In parallel, it caused lipid peroxidation and reactive oxygen species (ROS) formation and Mn overload. MZ inhibited superoxide dismutase and inducted catalase and glutathione S-transferase activities. Nitric oxide and reduced glutathione levels were significantly decreased by MZ. Heat shock proteins (HSP70 and HSP83) and Nrf2 mRNA levels were significantly augmented in MZ-exposed flies. Our study reinforced the use of Drosophila melanogaster as a reliable model for the study of biochemical targets of pesticides, and based on our data, MZ induced oxidative damage and Mn accumulation in a concentration-dependent manner. An adaptative cellular state was inducted by the lower concentration of pesticide, possibly contributing to the slighter damage observed.

Eugenia uniflora L. (Myrtaceae family), also known as “pitanga”, is a tree species widely used in popular medicine. Despite the well documented beneficial effects of the extracts and essential oils from this plant, little is known about its toxicity. We performed a phytochemical fingerprinting and evaluated the toxicity induced by the Eugenia uniflora leaves essential oil in a Drosophila melanogaster model. In order to understand the biochemical mechanisms involved in E. uniflora essential oil toxicity, changes in the Nrf2 signaling as well as the hallmarks of oxidative stress were measured. The exposure of adult flies to the essential oil via a fumigant method resulted in increased mortality and locomotor deficits. In parallel, an oxidative stress response signaling, evidenced by changes in ROS production, lipid peroxidation, alterations in the activity of antioxidant enzymes and expression of Nrf2 protein targets occurred. In the light of our findings, attention is drawn to the indiscriminate use of this plant for medicinal purposes. In addition, a potential bio-insecticidal activity of Eugenia uniflora volatile compounds is suggested, a fact that needs to be further explored.

Maternal obesity and metabolic diseases are two of the most important potential dangers to offspring, given that impaired offspring may cause deficiencies that impair the adult life and health. This study evaluated the oxidative damage, the enzymatic antioxidant defenses, and the enzymes of fatty acid metabolism, such as Acyl-CoA Synthetase and Acetyl-CoA Synthetase (mRNA expression levels), as well as the modulation of cell stress signaling pathway, as Hsp83, and gene expression and insulin-like peptide DILP6 in Drosophila melanogaster models that received a high fat diet (HFD) (10% and 20% of coconut oil) throughout their development period. After 7 days, the progenitor flies were removed and, the remaining eggs were monitored daily, until the eclosion. The descendants were then exposed to a regular diet (RD). The results revealed that the HFD caused a decrease in the proportion of eclosion, lifespan, MTT reduction in mitochondrial enriched fractions, AceCS1 levels, mRNA expression levels (SOD and CAT), and in catalase activity a decrease was only observed in the group that received the highest concentration of coconut oil. In parallel, it was demonstrated an increase in the upregulation of HSP83 mRNA levels, but only when 10% of coconut oil was added, and an increase in glucose and triglyceride levels, as well as in DILP6 mRNA levels in larger concentration of coconut oil tested (20%). In conclusion, flies that have progenitors fed with HFD can develop metabolic dysfunctions, causing oxidative insults, which are involved in the shortening of lifespan.

PTZ is a convulsive agent that acts via selective blockage of GABAA receptor channels, whereas 4-AP leads to a convulsive episode via blockage of K+ channels. However, the mechanism(s) by which pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) cause toxicity to Drosophila melanogaster needs to be properly explored, once it will help in establishing an alternative model for development of proper therapeutic strategies and also to counteract the changes associated with exposure to both epileptic drugs. For the purpose, we investigated the effects of exposure (48 h) to PTZ (60 mM) and/or 4-AP (20 mM) on survival, locomotor performance, and biochemical markers in the body and/or head of flies. 4-AP-fed flies presented a higher incidence of mortality and a worse performance in the open field test as compared to non-treated flies. 4-AP also caused a significant increase in the reactive species (RS) and protein carbonyl (PC) content in the body and head. Also a significant increase in catalase and acetylcholinesterase (AChE) activities was observed in the body. In the same vein, PTZ exposure resulted in a significant increase in RS, thiobarbituric acid reactive substances (TBARS), PC content, and catalase activity in the body. PTZ exposure also caused a significant increase in AChE activity both in body and head. It is important to note that PTZ-treated flies also down-regulated the NRF2 expression. Moreover, both 4AP- and PTZ-fed flies presented a significant decrease in MTT reduction, down-regulation, and inhibition of SOD in body. However, SOD was significantly more active in the head of both 4-AP and PTZ-treated flies. Our findings provide evidence regarding the toxicological potential of both PTZ and/or 4-AP to flies. This model will help in decoding the underlying toxicological mechanisms of the stated drugs. It will also help to properly investigate the therapeutic strategies and to counteract the drastic changes associated with both epileptogenic drugs.

Aims Many studies have been demonstrating the role of mitochondrial function in acetaminophen (APAP) hepatotoxicity. Since APAP is commonly consumed with caffeine, this work evaluated the effects of the combination of APAP and caffeine on hepatic mitochondrial bioenergetic function in mice. Main methods Mice were treated with caffeine (20 mg/kg, intraperitoneal (i.p.)) or its vehicle and, after 30 minutes, APAP (250 mg/kg, i.p.) or its vehicle. Four hours later, livers were removed, and the parameters associated with mitochondrial function and oxidative stress were evaluated. Hepatic cellular oxygen consumption was evaluated by high-resolution respirometry (HRR). Key findings APAP treatment decreased cellular oxygen consumption and mitochondrial complex activities in the livers of mice. Additionally, treatment with APAP increased swelling of isolated mitochondria from mice livers. On the other hand, caffeine administered with APAP was able to improve hepatic mitochondrial bioenergetic function. Treatment with APAP increased lipid peroxidation and reactive oxygen species (ROS) production and decreased glutathione levels in the livers of mice. Caffeine administered with APAP was able to prevent lipid peroxidation and the ROS production in mice livers, which may be associated with the improvement of mitochondrial function caused by caffeine treatment. Significance We suggest that the antioxidant effects of caffeine and/or its interactions with mitochondrial bioenergetics may be involved in its beneficial effects against APAP hepatotoxicity.

The adverse effects of the alga Prasiola crispa extract (PcE) were investigated in a fruit fly (Drosophila melanogaster) and cockroach (Nauphoeta cinerea) model. In flies, toxicity was assessed as mortality and biochemical alterations including acetylcholinesterase (AChE) activity and oxidative stress markers. The cardiotoxic action of PcE was also examined in a model of semi-isolated cockroach heart. The administration of PcE (2 mg/ml) to flies for 24 h resulted in a marked increase in mortality rate (7.6-fold rise compared to control). AChE activity, glutathione (GSH) levels, and hydroperoxide formation remained unchanged. Fly glutathione S-transferase (GST) and catalase (CAT) activity were significantly altered after PcE treatment. Fraction III (ethyl acetate) of PcE was significantly more toxic to flies compared to fractions I (methanol) and II (ethanol). A significant decrease was noted in cockroach semi-isolated heart function. The addition of 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB), an oxidizing agent, concomitant with the extract significantly blocked this effect, suggesting that reduced compounds may be involved in the cardiotoxic action produced by PcE. Our results show for the first time the adverse effects of PcE in two insect models, Drosophila melanogaster and Nauphoetacinerea. The insecticidal properties of PcE may be related to changes in important antioxidant/detoxifying systems, as well as to changes in insect cardiac function.

Organic and inorganic forms of mercury are highly neurotoxic environmental contaminants. The exact mechanisms involved in mercury neurotoxicity are still unclear. Oxidative stress appears to play central role in this process. In this study, we aimed to validate an insect-based model for the investigation of oxidative stress during mercury poisoning of lobster cockroach Nauphoeta cinerea. The advantages of using insects in basic toxicological studies include the easier handling, rapid proliferation/growing and absence of ethical issues, comparing to rodent-based models. Insects received solutions of HgCl2 (10, 20 and 40 mg L-1 in drinking water) for 7 d. 24 h after mercury exposure, animals were euthanized and head tissue samples were prepared for oxidative stress related biochemical determinations. Mercury exposure caused a concentration dependent decrease in survival rate. Cholinesterase activity was unchanged. Catalase activity was substantially impaired after mercury treatment 40 mg L-1. Likewise, GST had a significant decrease, comparing to control. Peroxidase and thioredoxin reductase activity was inhibited at concentrations of 20 mg L-1 and 40 mg L-1 comparing to control. These results were accompanied by decreased GSH levels and increased hydroperoxide and TBARS formation. In conclusion, our results show that mercuric compounds are able to induce oxidative stress signs in insect by modulating survival rate as well as inducing impairments on important antioxidant systems. In addition, our data demonstrates for the first time that Nauphoeta cinerea represents an interesting animal model to investigate mercury toxicity and indicates that the GSH and thioredoxin antioxidant systems plays central role in Hg induced toxicity in insects.

Senecio brasilienis (Spreng) Less., is a species native from Brazil, popularly known as "Maria mole", and known to induce hepatotoxicity due to its high content of Pyrrolizidine alkaloids. Despite its toxicity, this plant is widely used in Brazilian folk medicine. Considering the antagonizing effects described for S. brasiliensis, we describe here molecular markers involved in the toxicity of hydroalcoholic extract from leaves of S. brasiliensis (HESB) in Drosophila melanogaster. Phytochemical analysis of HESB revealed the presence of phenolic acids and flavonoids. A significant antioxidant potential against ABTS+ and DPPH radical was found in parallel. Ingestion of extract did not alter the survival and locomotor activity of adult flies. However when ingested along the larval developmental phase, the eclosion rate of flies was interrupted at higher concentration of extract. To comprehend this phenomenon several analysis were conducted in larvae. HESB stimulated activity of antioxidant enzymes SOD and GST, and increased GSH/GSSG ratio and ROS production. Additionally, HESB caused a significant decrease of cell viability. The mRNA expression of Nrf2, TrxR, CAT, Drice and Dilp6 were also significantly up-regulated. HESB caused significant decrease on the phosphorylation of MAPKs and AKT. In parallel, PARP cleavage and caspases 3/7 activity were stimulated. In addition, glucose, glycogen and triglycerides levels were decreased. Taken together our study depicts a disruption in the eclosion of D. melanogaster possibly attributed to the inhibition of kinases implied in developmental process, energetic demand and induction of apoptotic cell death process.

Physiopathological conditions such as acute liver failure (ALF) induced by acetaminophen (APAP) can often impair the mitochondrial bioenergetics. Diphenyl diselenide [(PhSe)2] has been shown protects against APAP-induced ALF. The present study aimed to clarify the signaling mechanism involved in the protection of bioenergetics dysfunction associated with ALF-induced by APAP overdose. Mice received APAP (600mg/kg) or (PhSe)2 (15.6mg/kg) alone, or APAP+(PhSe)2, all the solutions were administered by the intraperitoneal (i.p.). Samples of liver, blood and liver mitochondria were collected at 2 and 4h after APAP administration. APAP-induced ALF was able to induce ALF by means of alteration on liver injury biomarkers, increased Nitrite and Nitrate levels and the impairment of oxidative phosphorylation capacity (OXPHOS). In parallel, APAP overdose promoted activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and Heat shock protein 70 (HSP70) expression. (PhSe)2 was able to abolish the APAP-induced decline of OXPHOS and changes on the Nrf2-ARE pathway. In addition, (PhSe)2 elevated the levels of peroxisome proliferator-activated receptor-γ coactivator (PGC-1α), helping to restore the levels of nuclear respiratory factor 1 (NRF1) associated with mitochondrial biogenesis. In summary, the treatment with (PhSe)2 maintained mitochondrial function, promoted genes related to mitochondrial dynamic and demonstrating to play critical role in the modulation of cellular protective responses during ALF.

Croton campestris A.St.-Hil. (Euphorbiaceae) is a species native to Northeast Brazil used by traditional communities for the treatment of a variety of health problems. However, potential toxicological effects of this plant are unknown.The potential toxicity of the hydroalcoholic extract of C. campestris leaves on Drosophila melanogaster insect model, additionally with phytochemical constitution and cellular mechanisms mediating the action of extract were analysed in this study.Constituents of the extract were evaluated by HPLC. In vitro antioxidant potential of extract was analysed by DPPH, ABTS and FRAP. Flies injected culture medium mixed with extract (0.1-50 mg/mL) for 72 h. After, ROS production was evaluated by DCF-DA oxidation. Phosphorylation of MAPK signalling pathway was investigated by Western blotting method. Activity of antioxidant enzymes was analysed in homogenates.Major components of the extract include quercetin (38.11 ± 0.06 mg/g), caffeic acid (20.06 ± 0.17 mg/g) and kaempferol (15.45 ± 0.05 mg/g). Consumption of the extract impaired locomotor performance and induced fly death of flies (LCOur data show important toxicological effects of C. campestris leading to increased mortality and impaired locomotor performance accompanied by induction of cell stress markers in flies. The study draws attention to the indiscriminate use of plant extracts.

The consumption of a high-fat diet (HFD) causes alteration in normal metabolism affecting lifespan of flies; however molecular mechanism associated with this damage in flies is not well known. This study evaluates the effects of ingestion of a diet supplemented with 10% and 20% of coconut oil, which is rich in saturated fatty acids, on oxidative stress and cells stress signaling pathways. After exposure to the diet for seven days, cellular and mitochondrial viability, lipid peroxidation and antioxidant enzymes SOD and CAT activity, and mRNA expression of antioxidant enzymes HSP83 and MPK2 were analyzed. To confirm the damage effect of diet on flies, survival and lifespan were investigated. The results revealed that the HFD augmented the rate of lipid peroxidation and SOD and CAT activity and induced a higher expression of HSP83 and MPK2 mRNA. In parallel, levels of enzymes involved in lipid metabolism (ACSL1 and ACeCS1) were increased. Our data demonstrate that association among metabolic changes, oxidative stress, and protein signalization might be involved in shortening the lifespan of flies fed with a HFD.

We evaluated the activity and expression of antioxidant enzymes in the cerebellum and cortex of Swiss adult male mice exposed to methylmercury (MeHg) in drinking water (40mg/L) during 21 days. The activity of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were determined spectrophotometrically. The expression (protein levels) of GPx1 and GPx4 isoforms, TrxR1 as well as heat shock protein 70 (HSP70) were evaluated using specific antibodies and normalized by actin levels. The exposure of mice to MeHg caused a significant impairment in locomotors performance in the open field test (crossings and rearing). This result was followed by a significant reduction of GPx and TrxR activities in the cerebellum and cortex when compared to untreated animals. We also observed a substantial decrease in GPx1, GPx4 and TrxR1 protein levels in the cerebellum, while in the cerebral cortex, only GPx4 and TrxR1 were decreased after MeHg treatment. The activities of the antioxidant enzymes GR, GST, CAT and SOD were increased in the cerebellum after MeHg administration to mice. In contrast, only CAT was increased in the cerebral cortex of MeHg-treated animals. The expression of HSP70 was up-regulated only in the cerebellum where MeHg-exposed mice showed a significant increase in the immunocontent of HSP70 when compared to controls. This is the first report showing a role for GPx4 in the neurotoxicity induced by MeHg in vivo. In addition, our data indicates that the selenoproteins GPx and TrxR as main targets during MeHg exposure, which may be considered in biomarker studies.

In this study, we investigated the effect of diphenyl ditelluride (PhTe)(2) administration (10 and 50 μmol/kg) on adult mouse behavioral performance as well as several parameters of oxidative stress in the brain and liver. Adult mice were injected with (PhTe)(2) or canola oil subcutaneously (s.c.) daily for 7 days. Results demonstrated that (PhTe)(2) induced prominent signs of toxicity (body weight loss), behavioral alterations and increased in lipid peroxidation in brain. 50 μmol/kg (PhTe)(2) inhibited blood δ-aminolevulinic acid dehydratase (δ-ALA-D), a redox sensitive enzyme. (PhTe)(2) caused an increase in cerebral non-protein thiol (NPSH) and protein thiol (PSH) groups. In the liver, 50 μmol/kg (PhTe)(2) decreased NPSH, but did not alter the content of protein thiol groups. (PhTe)(2) decreased cerebral antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), and thioredoxin reductase (TrxR). In liver, (PhTe)(2) increase SOD and GR and decreased GPx activity. Results obtained herein suggest that the brain was more susceptible to oxidative stress induced by (PhTe)(2) than the liver. Furthermore, we have demonstrated for the first time that TrxR is an in vivo target for (PhTe)(2.) Combined, these results highlight a novel molecular mechanism involved in the toxicity of (PhTe)(2). In particular the inhibition of important selenoenzymes (TrxR and GPx) seems to be involved in the neurotoxicity associated with (PhTe)(2) exposure in adult mice.

The heavy metal mercury is a known toxin, but while the mechanisms involved in mercury toxicity have been well demonstrated in vertebrates, little is known about toxicological effects of this metal in invertebrates. Here, we present the results of our study investigating the effects associated with exposure of fruit fly Drosophila melanogaster to inorganic mercury (HgCl2 ). We quantify survival and locomotor performance as well as a variety of biochemical parameters including antioxidant status, MAPK phosphorylation and gene expression following mercury treatment. Our results demonstrate that exposure to Hg(II) through diet induced mortality and affected locomotor performance as evaluated by negative geotaxis, in D. melanogaster. We also saw a significant impact on the antioxidant system including an inhibition of acetylcholinesterase (Ache), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities. We found no significant alteration in the levels of mRNA of antioxidant enzymes or NRF-2 transcriptional factor, but did detect a significant up regulation of the HSP83 gene. Mercury exposure also induced the phosphorylation of JNK and ERK, without altering p38(MAPK) and the concentration of these kinases. In parallel, Hg(II) induced PARP cleavage in a 89 kDa fragment, suggesting the triggering of apoptotic cell death in response to the treatment. Taken together, this data clarifies and extends our understanding of the molecular mechanisms mediating Hg(II) toxicity in an invertebrate model.

Gender is one of the most important factors in mammalian development and response to exogenous agents. Although there is increasing evidence that health effects of toxic xenobiotics differ in prevalence or are manifested differently in male and female, the molecular mechanisms related to these events remain unclear. In order to investigate the possible influence of gender, male and female Wistar rats from the same litter were exposed to zinc chloride (5 mg/kg, i.p.) or malathion (250 mg/kg, i.p.) 24 hr prior to the analyses of biochemical parameters related to the cholinergic and glutathione-antioxidant systems in cerebral cortex and hippocampus. After treatments, acetylcholinesterase (AChE) activity was reduced in the hippocampus and cerebral cortex of male and female rats treated with malathion, but the effect was more pronounced in the male group. Glutathione reductase (GR) and γ-glutamyl-transpeptidase activities were reduced in the hippocampus of males and females and a gender-specific effect of malathion was seen for glutathione S-transferase (GST), which was decreased only in male hippocampus and cortex, and for male cortical GR. Zinc chloride treatment decreased AChE activity in male and female cerebral cortex, with no obvious effect in the hippocampus. Male-specific antioxidant-related enzyme activity reductions were seen after zinc treatment for cortical GR, GST, glutathione peroxidase and glucose-6-phosphate dehydrogenase; and hippocampal GR. These results clearly demonstrate a greater detrimental effect on antioxidant-related enzyme activities in male hippocampus and cerebral cortex when rats were acutely exposed to malathion and zinc, demonstrating that the research on gender-related differences in health effects caused by xenobiotic and/or essential elements requires further attention.

Diphenyl diselenide (PhSe)(2) is a synthetic organoselenium compound displaying glutathione peroxidase-like activity. Protective and antioxidant potential of (PhSe)(2) have been extensively investigated in in vivo and in vitro studies. In spite of this, there is a lack of studies addressed to the investigation of potential cytotoxic effect and signaling pathways modulated by this compound. Herein, we aimed to analyze the effects of 24-h treatment with (PhSe)(2) on cell viability and a possible modulation of signaling pathways in human neuroblastoma cell line SH-SY5Y. For this purpose, cells were incubated with (PhSe)(2) (0.3-30 μM) for 24 h and cell viability, apoptotic cell death and modulation of MAPKs (ERK1/2 and p38(MAPK)), and PKC substrates phosphorylation was determined. (PhSe)(2) treatment significantly decreased cell viability and increased the number of apoptotic cells with induction of PARP cleavage. An increase in ERK1/2 phosphorylation was observed at (PhSe)(2) 3 μM. In contrast, higher concentrations of the chalcogenide inhibited ERK1/2, p38(MAPK) and PKC substrate phosphorylation. Pre-treatment with ERK1/2 inhibitor, U0126, increased cell susceptibility to (PhSe)(2). Together, these data indicate a cytotoxic potential of (PhSe)(2) in a neuronal cell line, which appears to be mediated by the ERK1/2 pathway.

Methylmercury (MeHg) can cause deleterious effects in vertebrate tissues, particularly in the central nervous system. MeHg interacts with sulfhydryl groups from low and high molecular weight thiols in the blood, which can facilitate MeHg uptake into different tissues. The purpose of this study was to examine the effect of MeHg-Cysteine (MeHg-Cys) complex administration on Hg-uptake in cerebral areas (cortex and cerebellum), liver and kidney of adult mice. Animals were divided into four groups: control (1 mL/kg distilled water), MeHg (2 mg/kg), Cys (2 mg/kg) and MeHg-Cys complex (0.8 molar ratio). Mice received one intraperitoneal injection per day for 60 consecutive days. Treatment with MeHg significantly increased mercury concentrations in all tissues analysed when compared with the control group. The accumulation of mercury in brain and in liver was further increased in animals that received MeHg-Cys complex when compared with the MeHg alone group. However, renal Hg decreased in MeHg-Cys treated mice, when compared with the group treated only with MeHg. In summary, the transport of MeHg-Cys complex was tissue-specific, and we observed an increase in its uptake by liver and brain as well as a decrease in kidney.

Joinville is an important industrial city in Santa Catarina, southern Brazil, and also a risk factor for the Babitonga drainage basin. Oxidative stress-related parameters were evaluated in caged tilapia (Oreochromis niloticus) exposed for 7 days (sites S1 and S2) in a Babitonga drainage basin tributary river. Site S1 showed enhanced levels of hepatic CYP1A, CYP2B-like and glutathione S-transferase activity, while site S2 showed decreased levels of glutathione and increased lipoperoxidation indexes, catalase, glutathione peroxidase and glutathione reductase activity. Correlation analyses revealed that oxidative stress-related parameters behaved like a group of interrelated variables, while CYPs and glutathione S-transferase seem to be independent. New putative biomarkers were evaluated in the tilapia brain. Caspase-3 activation (both sites), decreased in p38MAPK phosphorylation (site S2) and decreased expression in HSP70 (site S1) were observed. Data indicate that employed variables, when used as a group (oxidative stress-related parameters, CYP1A/2B-like, caspase-3, HSP70 and protein kinases) can be useful as predictors of pollution.

Brocardo PS, Budni J, Pavesi E, Franco JL, Uliano-Silva M, Trevisan R, Terenzi MG, Dafre AL, Rodrigues ALS. Folic acid administration prevents ouabain-induced hyperlocomotion and alterations in oxidative stress markers in the rat brain. Bipolar Disord 2010: 12: 414–424. © 2010 The Authors. Journal compilation © 2010 John Wiley & Sons A/S. Objective: Bipolar disorder (BD) is a chronic, prevalent, and highly debilitating psychiatric illness. Folic acid has been shown to have antidepressant-like effects in preclinical and clinical studies and has also been suggested to play a role in BD. The present work investigates the therapeutic value of folic acid supplementation in a preclinical animal model of mania induced by ouabain. Methods: Male Wistar rats were treated twice daily for seven days with folic acid (10, 50, and 100 mg/kg, p.o.) or the mood stabilizer lithium chloride (LiCl) (45 mg/kg, p.o.). One day after the last dose was given, the animals received an i.c.v. injection of ouabain (10 μM), a Na+,K+-ATPase-inhibiting compound. Locomotor activity was assessed in the open-field test. Thiobarbituric acid-reactive substance (TBARS) levels, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were measured in the cerebral cortex and hippocampus. Results: Ouabain (10 μM, i.c.v.) significantly increased motor activity in the open-field test, and seven days of pretreatment with folic acid (50 mg/kg, p.o.) or LiCl (45 mg/kg, p.o.) completely prevented this effect. Ouabain treatment elicited lipid peroxidation (increased TBARS levels) and reduced GPx activity in the hippocampus. GR activity was decreased in the cerebral cortex and hippocampus. These effects were prevented by pretreatment with folic acid and LiCl. Conclusions: Our results show that folic acid, similarly to LiCl, produces a clear antimanic action and prevents the neurochemical alterations indicative of oxidative stress in an animal model of mania.

A beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis, order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS-PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 x 10(-6) micromol/(min x mg) were found for this enzyme, determined by p-nitrophenyl-beta-D: -hexosaminide substrate digestion. Optimal pH and temperature for beta-N-Acetylhexosaminidase activity were 5.0 and 60 degrees C, respectively. Enzyme activity was inhibited by sodium selenate (Na(2)SeO(4)), mercuric chloride (HgCl(2)) and sodium dodecyl sulfate (C(12)H(25)SO(4)Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the beta-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.

Introduction and Objectives: In the present study, we aimed to investigate the toxic effects of Prasiola crispa extract on a fruit fly (Drosophila melanogaster) model. Methods: Toxicity was assessed as % mortality, negative geotaxis behavior and acetylcholine esterase (AchE), glutathione S-transferase (GST), catalase (CAT) activities as well as glutathione content (GSH) and hydroperoxide formation. Results and discussion: Administration of algae extract (2 mg/ml) to flies for 24 hours resulted in a massive increase in mortality (760 %, compared to control). It was also observed a significant increase in climbing performance, indicating an alteration in negative geotaxis behavior. The AchE activity was unchanged after algae extract treatment for 24 hours. However, GST activity was significantly increased after Prasiola crispa administration. The CAT activity was significantly decreased in flies that received algae extract for 24 hours. Glutathione levels and hydroperoxide formation remained unchanged. Our results show for the first time the toxic effects of an Antarctic algae extract in Drosophila melanogaster. Conclusion: The insecticide action of Prasiola crispa may be related to changes on vital antioxidant systems. Further studies are necessary to elucidate the exact mechanisms of toxicity of this Antarctic algae to Drosophila melanogaster.

We investigated the antidepressant-like effect of zinc chloride (zinc) administered acutely during 7 days (i.p. route), or chronically during 30 days (oral route) in the forced swimming test (FST) in rats. It was also investigated whether the antidepressant-like effect of zinc is associated with changes in the glutathione antioxidant system in the Wistar rat brain. Animals receiving a single zinc dose (5, 15 and 30 mg/kg, i.p.) 24 h prior to analysis showed no changes in the FST, but glutathione reductase and glutathione S-transferase activity were reduced in the hippocampus and cerebral cortex. This treatment did not, however, affect the glutathione status (GSH and GSSG) in both brain structures. The 7-day zinc treatment (1, 5 and 15 mg/kg, i.p.) caused a mild though significant antidepressant-like effect in the FST at the highest dosing, without affecting the glutathione antioxidant system. Finally, a consistent antidepressant-like effect was achieved in the FST after chronic (30 days) zinc treatment (300 mg/L, p.o.). This was accompanied by a significant increase in total glutathione levels in the hippocampus and cerebral cortex. The good response to oral treatment in the FST led us to investigate other variables, such as ERK phosphorylation and BDNF expression. Similar to therapeutic antidepressants, zinc in chronic oral treatment produced an increase in ERK phosphorylation and BDNF expression in the cerebral cortex. It is our hypothesis that up-regulation of neuroprotective effectors (GSH, ERK and BDNF) may be related to the antidepressant properties of zinc, but this will require additional work to be confirmed.

Glutathione depletion is a key factor in the development of acute pancreatitis. Our aim was to study the regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, in edematous or necrotizing pancreatitis in rats. Glutathione levels were kept low in necrotizing pancreatitis for several hours, with no increase in protein or mRNA levels of glutamate cysteine ligase subunits, despite binding of RNA polymerase II to their promoters and coding regions. The survival signal pathway mediated by ERK and c-MYC was activated, and c-MYC was recruited to the promoters. The failure in gene up-regulation seems to be due to a marked increase in cytosolic ribonuclease activity. In contrast, in edematous pancreatitis glutathione levels were depleted and rapidly restored, and protein and mRNA expression of glutamate cysteine ligase increased markedly due to enhanced transcription mediated by recruitment of c-MYC, NF-kappaB, and SP-1 to the promoters. No increase in cytosolic ribonuclease activity was found in this case. We propose a novel pathophysiological mechanism to differentiate necrotizing from edematous pancreatitis, which is the inefficient up-regulation of glutamate cysteine ligase caused by increased cytosolic ribonuclease activity in the severe form of the disease. This mechanism would abrogate a rapid recovery of glutathione levels.

The present study aimed at investigating the potential in vitro protective effect of the organochalcogenide diphenyl diselenide - (PhSe)2 - against hydrogen peroxide (H2O2)-induced toxicity in rat hippocampal slices. Hippocampal slices were treated for 1 h with H2O2 (2 mM) in the presence or absence of (PhSe)2 (0.1-10 microM). H2O2 treatment significantly decreased cell viability (measured by MTT test) and the co-incubation with (PhSe)(2) (10 microM) significantly blunted such phenomenon. The non permeable thiol compounds dithiothreitol (DTT) (100 microM) or reduced glutathione (GSH) (100 microM), which did not display protective effects against H2O2-induced loss of cell viability per se, significantly improved the protective effects elicited by (PhSe)2. Conversely, the permeable form of GSH (GSH monoethyl ester) was unable to alter the neuroprotection mediated by (PhSe)2. The treatment of rat hippocampal slices with H2O2 also increased the lipid peroxidation and decreased the intracellular GSH levels. Moreover, (PhSe)2 (from 0.1 microM) significantly decreased H2O2-induced lipid peroxidation. Interestingly, H2O2 decreased GSH levels and this phenomenon was partially prevented by (PhSe)2. The potential effects of H2O2 on MAPKs phosphorylation (ERK1/2, p38 MAPK and JNK1/2) were also evaluated. Even though H2O2 (2 mM) did not alter p38 MAPK and JNK1/2 phosphorylation in hippocampal slices, it stimulated ERK1/2 phosphorylation and the co-incubation with (PhSe)2 (10 microM) blocked this effect. Taken together, the present results indicate that (PhSe)2 exerts protective effects against H2O2-induced oxidative damage in hippocampal slices and avoided the increase in ERK1/2 phosphorylation promoted by H2O2. The neuroprotective effect of compound seems to be related to its thiol-peroxidase-like activity and appears to occur at the extracellular milieu because a permeable form of GSH was unable to improve the protective effect of the compound as did the impermeable GSH.

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm(2) pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract. Results The extract at concentrations of 250 and 125 μg/mL inhibited 80.39 and 54.27% of promastigote (LC50 value = 105.78 μg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC50 value = 110.69 μg/mL) and against epimastigote (LC50 value = 245.23 μg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 μg/mL with 95.41% (LC50 value = 141.01 μg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (

Brain preconditioning refers to a wide range of treatments that induce a neuronal tolerance state where neuronal tissue become more resistant to a subsequent lethal insult. The mechanisms underlying the preconditioning-induced brain tolerance are not fully understood, but up-regulation of antioxidant enzymes activity has been suggested to play an important role. In order to test this hypothesis, evaluation of glutathione (GSH) scavenger system was carried out in mice showing the neuroprotective effect of NMDA preconditioning against quinolinic acid (QA)-induced seizures. NMDA is known to prevent seizures in 53% of the animals and completely prevent neural damage against QA. Mice were preconditioned by a non-convulsant NMDA dose (75 mg/kg, 10 ml/kg i.p.) 24 h before QA infusion (4 microl, 9.2 mM i.c.v.). GSH content and enzymatic activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) were evaluated in the cerebral cortex and hippocampus 24 h after QA infusion. NMDA preconditioning and QA infusion did not alter GSH content, GR and G6PDH activities, however, an increase in GST activity was observed in the cerebral cortex from mice. Moreover, NMDA pretreatment was able to prevent the QA-induced decrease in hippocampal GPx activity, but it was not effective against the decreased cortical GPx activity. These results indicate that, although NMDA preconditioning and QA toxicity modulate the activity of some GSH related enzymes, GSH metabolism is not directly linked to the neuroprotective effect induced by NMDA preconditioning.

Chelating therapy has been reported as a useful approach for counteracting mercurial toxicity. Moreover, 2,3-dimercapto-1-propanesulfonic acid (DMPS), a tissue-permeable metal chelator, was found to increase urinary mercury excretion and decrease mercury content in rat brain after methylmercury (MeHg) exposure. We evaluated the capability of DMPS to reduce MeHg-induced motor impairment and cerebellar toxicity in adult mice. Animals were exposed to MeHg (40 mg/L in drinking water, ad libitum ) during 17 days. In the last 3 days of exposure (days 15–17), animals received DMPS injections (150 mg/kg, i.p.; once a day) in order to reverse MeHg-induced neurotoxicity. Twenty-four hours after the last injection (day 18), behavioral tests related to the motor function (open field and rotarod tasks) and biochemical analyses on oxidative stress-related parameters (cerebellar glutathione, protein thiol and malondyaldehyde levels, glutathione peroxidase and glutathione reductase activities) were carried out. Histological analyses for quantifying cellular damage and mercury deposition in the cerebellum were also performed. MeHg exposure induced a significant motor deficit, observed as decreased locomotor activity in the open field and decreased falling latency in the rotarod apparatus. DMPS treatment displayed an ameliorative effect toward such behavioral parameters. Cerebellar glutathione and protein thiol levels were not changed by MeHg or DMPS treatment. Conversely, the levels of cerebellar thiobarbituric acid reactive substances (TBARS), a marker for lipid peroxidation, were increased in MeHg-exposed mice and DMPS administration minimized such phenomenon. Cerebellar glutathione peroxidase activity was decreased in the MeHg-exposed animals, but DMPS treatment did not prevent such event. Histological analyses showed a reduced number of cerebellar Purkinje cells in MeHg-treated mice and this phenomenon was completely reversed by DMPS treatment. A marked mercury deposition in the cerebellar cortex was observed in MeHg-exposed animals (granular layer > Purkinje cells > molecular layer) and DMPS treatment displayed a significant ameliorative effect toward these phenomena. These findings indicate that DMPS displays beneficial effects on reversing MeHg-induced motor deficits and cerebellar damage in mice. Histological analyses indicate that these phenomena are related to its capability of removing mercury from cerebellar cortex.

Cipura paludosa (Iridaceae), a native plant widely distributed in the north of Brazil, is used in traditional medicine as an anti-inflammatory and analgesic agent, against tuberculosis and gonorrhoea and for regulation of menstrual flow. However, scientific studies on the pharmacological properties of C. paludosa are scarce. We have examined the potential protective effects of the ethanolic extract of C. paludosa against methyl mercury (MeHg)-induced neurotoxicity in adult mice. MeHg was diluted in drinking water (40 mg/l, freely available) and the ethanolic C. paludosa extract (CE) was diluted in a 150 mM NaCl solution and administered by gavage (10 and 100 mg/kg body weight, respectively, twice a day). Because treatment lasted for 14 days and each animal weighed around 40 g, the total dosage of plant extract given to each mouse was 5.6 and 56 g, respectively. After the treatment period, MeHg exposure induced a significant deficit in the motor coordination, which was evident by a reduction (90%) in the falling latency in the rotarod apparatus. Interestingly, this phenomenon was completely recovered to control levels by CE co-administration, independent of dosages. MeHg exposure inhibited cerebellar glutathione peroxidase (mean percentage inhibition of 42%) – an important enzyme involved in the detoxification of endogenous peroxides – and this effect was prevented by co-administration of CE. Conversely, MeHg exposure increased cerebellar glutathione reductase activity (mean percentage inhibition of 70%), and this phenomenon was not affected by C. paludosa co-administration. Neither MeHg nor CE changed the cerebellar glutathione levels. This study has shown for the first time, the in vivo protective effects of CE against MeHg-induced neurotoxicity. In addition, our findings encourage studies concerning the beneficial effects of C. paludosa on neurological conditions related to excitotoxicity and oxidative stress.