Acute embryonic exposure of zebrafish to permethrin induces behavioral changes related to anxiety and aggressiveness in adulthood.

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.

Honey protects against wings posture error and molecular changes related to mitochondrial pathways induced by hypoxia/reoxygenation in adult Drosophila melanogaster.

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.

Data on the phosphorylation of p38MAPK and JNK induced by chlorpyrifos in Drosophila melanogaster.

Exposure to organophosphate compounds, such as chlorpyrifos, has been linked to disturbances on cell signaling pathways. Mitogen activated protein kinases (MAPK) are a family of protein kinases involved in a range of cellular processes, including stress response, apoptosis and survival. Therefore, changes in the activation state of these kinases may characterize key mechanisms of toxicity elicited by xenobiotics. Here we report data on the phosphorylation of p38MAPK and JNK, members of the MAPK family, in Drosophila melanogaster exposed to chlorpyrifos, as characterized by western blotting assays.

Brazilian Pampa Biome Honey Protects Against Mortality, Locomotor Deficits and Oxidative Stress Induced by Hypoxia/Reperfusion in Adult Drosophila melanogaster.

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), NFκß, 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.

Oxidative stress markers in fish (Astyanax sp. and Danio rerio) exposed to urban and agricultural effluents in the Brazilian Pampa biome.

Aquatic ecosystems are under constant risk due to industrial, agricultural, and urban activities, compromising water quality and preservation of aquatic biota. The assessment of toxicological impacts caused by pollutants to aquatic environment using biomarker measurements in fish can provide reliable data to estimate sublethal effects posed by chemicals in contaminated areas. In this study, fish (Astyanax sp. and Danio rerio) exposed to agricultural and urban effluents at the Vacacaí River, Brazil, were tested for potential signs of aquatic contamination. This river comprehends one of the main watercourses of the Brazilian Pampa, a biome with a large biodiversity that has been neglected in terms of environmental and social-economic development. Sites S1 and S2 were chosen by their proximity to crops and wastewater discharge points, while reference site was located upstream of S1 and S2, in an apparently non-degraded area. Fish muscle and brain tissues were processed for determination of acetylcholinesterase as well as oxidative stress-related biomarkers. The results showed signs of environmental contamination, hallmarked by significant changes in cholinesterase activity, expression of metallothionein, antioxidant enzymes, glutathione levels, and activation of antioxidant/cell stress response signaling pathways in fish exposed to contaminated sites when compared to reference. Based on these results, it is evidenced that urban and agricultural activities are posing risk to the environmental quality of water resources at the studied area. It is also demonstrated that cell stress biomarkers may serve as important tools for biomonitoring and development of risk assessment protocols in the Pampa biome.

Effects of Hg(II) exposure on MAPK phosphorylation and antioxidant system in D. melanogaster.

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.

A Study on the Quality and Identity of Brazilian Pampa Biome Honey: Evidences for Its Beneficial Effects against Oxidative Stress and Hyperglycemia.

We characterized, for the first time, the quality and identity of Brazilian Pampa biome honey and its antioxidant properties in vitro (FRAP, DDPH and ABTS). The potential protective effect of honey against oxidative stress induced by iron (Fe) and paraquat, (PQ) in a Drosophila melanogaster model (in vivo) was also tested. The results indicated that all honey samples tested showed antioxidant activity in vitro. Flies treated with honey showed increased lifespan and were protected against oxidative stress induced by Fe and PQ. Despite the high concentration of sugars in honey (approximately 70-80%), our results demonstrate a hypoglycemic-like effect of honey in Drosophila. Thus, this study demonstrates the high quality of Brazilian Pampa biome honey as well as its significant antioxidant activity in vitro and in vivo, pointing to the potential use of this natural product as an alternative in the therapy of oxidative stress-associated diseases.

Is the lobster cockroach Nauphoeta cinerea a valuable model for evaluating mercury induced oxidative stress?

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 40mgL(-1) in drinking water) for 7d. 24h 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 40mgL(-1). Likewise, GST had a significant decrease, comparing to control. Peroxidase and thioredoxin reductase activity was inhibited at concentrations of 20mgL(-1) and 40mgL(-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.

Evidences for a role of glutathione peroxidase 4 (GPx4) in methylmercury induced neurotoxicity in vivo.

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.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8 percent inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions.

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3'3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3'3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Ebselen and diphenyl diselenide change biochemical hepatic responses to overdosage with paracetamol.

The toxicity of paracetamol is largely related to its conversion to the reactive intermediate alkylating metabolite N-acetyl-para-benzo-quinoneimine (NAPQI). δ-Aminolevulinate dehydratase (δ-ALA-D) is a sulfhydril containing enzyme which is extremely sensitive to oxidizing and alkylating agents. In the present study, we examined whether acute treatment with paracetamol changes δ-ALA-D activity. The influence of two organochalcogenides with glutathione peroxidase-like activity, diphenyl diselenide [(PhSe)(2)] and ebselen was also assessed as potential protecting agents against paracetamol toxicity. Paracetamol (1200mg/kg for three days 4h after the injection of DMSO, diphenyl diselenide (100µmol/kg) or ebselen (100µmol/kg) caused an inhibition of about 40% (P < 0.01) in hepatic δ-ALA-D. Ebselen restored enzyme activity to control values. Non-protein-SH and ascorbic acid were diminished to 50% of control value by paracetamol, independent of chalcogenides treatment (all P values <0.05). In view of the fact that paracetamol caused a massive reduction in non-protein-SH and ascorbic acid, we realize that the protective effect of ebselen on δ-ALA-D activity is mediated by its thiol peroxidase-like activity or by a direct interaction with NAPQI and other reactive species formed during paracetamol metabolism.

Ebselen protects Ca2+ influx blockage but does not protect glutamate uptake inhibition caused by Hg2+.

The goal of this study was to investigate the isolated and combined effect of ebselen and Hg2+ on calcium influx and on glutamatergic system. We examined the in vitro effects of 2 phenyl-1,2-benzisoselenazol-3(2H)-ona), (Ebselen) on 45Ca2+ influx in synaptosomes of rat at rest and during depolarization and glutamate uptake into synaptosomes. Entry of 45Ca was measured during exposure to mercury in non-depolarizing and depolarizing solutions. Ebselen abolished the inhibition of 45Ca2+ influx on non-depolarizing conditions; however, ebselen did no modify inhibition uptake of 45Ca2+ caused by Hg2+ in high K+ depolarizing medium. Ebselen did not modify glutamate uptake inhibition caused by Hg2+ in synaptosomes. These results indicate that ebselen has an in vitro protective effect against Hg2+ induced inhibition of Ca2+ influx into synaptosomes, depending on the depolarizing conditions of the assay. The effects of Hg2+ on glutamate uptake were not modified by ebselen, suggesting that its protection is dependent on the target protein considered.