Ana Paula Pegoraro Zemolin

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 p38MAPK 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.

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.

Drosophila melanogaster - an embryonic model for studying behavioral and biochemical effects of manganese exposure

Embryonic animals are especially susceptible to metal exposure. Manganese (Mn) is an essential element, but in excess it can induce toxicity. In this study we used Drosophila melanogaster as an embryonic model to investigate biochemical and behavioral alterations due to Mn exposure. Flies were treated with standard medium supplemented with MnCl2 at 0.1 mM, 0.5 mM or 1 mM from the egg to the adult stage. At 0.5 mM and 1 mM Mn, newly ecloded flies showed significantly enhanced locomotor activity when assessed by negative geotaxis behavior. In addition, a significant increase in Mn levels (p < 0.0001) was observed, while Ca, Fe, Cu, Zn and S levels were significantly decreased. A significant drop in cell viability occurred in flies exposed to 1 mM Mn. There was also an induction of reactive oxygen species at 0.5 mM and 1 mM Mn (p < 0.05). At 1 mM, Mn increased Catalase (p < 0.005), Superoxide Dismutase (p < 0.005) and Hsp83 (p < 0.0001) mRNA expression, without altering Catalase or Superoxide Dismutase activity; the activity of Thioredoxin reductase and Glutatione-S-transferase enzymes was increased. Mn treatment did not alter ERK or JNK1/2 phosphorylation, but at 1 mM caused an inhibition of p38MAPK phosphorylation. Together these data suggest mechanisms of adaptation in the fly response to Mn exposure in embryonic life.

Effects of Bauhinia forficata Tea on Oxidative Stress and Liver Damage in Diabetic Mice.

This study was designed to evaluate the effects of Bauhinia forficata Link 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, and δ-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.

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.

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.

High-Fat Diet Induces Oxidative Stress and MPK2 and HSP83 Gene Expression in Drosophila melanogaster

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.

Sub-acute administration of ( S )-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate induces toxicity and oxidative stress in mice: unexpected effects of N-acetylcysteine

The organic tellurium compound (S)-dimethyl 2-(3-(phenyltellanyl) propanamide) succinate (TeAsp) exhibits thiol-peroxidase activity that could potentially offer protection against oxidative stress. However, data from the literature show that tellurium is a toxic agent to rodents. In order to mitigate such toxicity, N-acetylcysteine (NAC) was administered in parallel with TeAsp during 10 days. Mice were separated into four groups receiving daily injections of (A) vehicle (PBS 2.5 ml/kg, i.p. and DMSO 1 ml/kg, s.c.), (B) NAC (100 mg/kg, i.p. and DMSO s.c.), (C) PBS i.p. and TeAsp (92.5 μmol/kg, s.c), or (D) NAC plus TeAsp. TeAsp treatment started on the fourth day. Vehicle or NAC-treated animals showed an increase in body weight whereas TeAsp caused a significant reduction. Contrary to expected, NAC co-administration potentiated the toxic effect of TeAsp, causing a decrease in body weight. Vehicle, NAC or TeAsp did not affect the exploratory and motor activity in the open-field test at the end of the treatment, while the combination of NAC and TeAsp produced a significant decrease in these parameters. No DNA damage or alterations in cell viability were observed in leukocytes of treated animals. Treatments produced no or minor effects on the activities of antioxidant enzymes catalase, glutathione peroxidase and glutathione reductase, whereas the activity of the thioredoxin reductase was decreased in the brain and increased the liver of the animals in the groups receiving TeAsp or TeAsp plus NAC. In conclusion, the toxicity of TeAsp was potentiated by NAC and oxidative stress appears to play a central role in this process.

Reproductive dysfunction after mercury exposure at low levels: evidence for a role of glutathione peroxidase (GPx) 1 and GPx4 in male rats

Mercury is a ubiquitous environmental pollutant and mercury contamination and toxicity are serious hazards to human health. Some studies have shown that mercury impairs male reproductive function, but less is known about its effects following exposure at low doses and the possible mechanisms underlying its toxicity. Herein we show that exposure of rats to mercury chloride for 30 days (first dose 4.6µgkg-1, subsequent doses 0.07µgkg-1day-1) resulted in mean (±s.e.m.) blood mercury concentrations of 6.8±0.3ngmL-1, similar to that found in human blood after occupational exposure or released from removal of amalgam fillings. Even at these low concentrations, mercury was deposited in reproductive organs (testis, epididymis and prostate), impaired sperm membrane integrity, reduced the number of mature spermatozoa and, in the testes, promoted disorganisation, empty spaces and loss of germinal epithelium. Mercury increased levels of reactive oxygen species and the expression of glutathione peroxidase (GPx) 1 and GPx4. These results suggest that the toxic effects of mercury on the male reproductive system are due to its accumulation in reproductive organs and that the glutathione system is its potential target. The data also suggest, for the first time, a possible role of the selenoproteins GPx1 and GPx4 in the reproductive toxicity of mercury chloride.