Antonio Batista Pereira

Freezing and desiccation injury resistance in the filamentous green alga Klebsormidium from the Antarctic, Arctic and Slovakia

The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aeroterrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic — South Shetlands, King George Island, Arctic — Ellesmere Island, Svalbard, Central Europe — Slovakia) were studied. Each strain was exposed to several freezing (−4°C, −40°C, −196°C) and desiccation (+4°C and + 20°C) regimes, simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries. Freezing down to −196°C was the most harmful regime for all studied strains. Freezing at −4°C did not influence the survival of studied strains. Further, freezing down to −40°C (at a speed of 4°C/min) was not fatal for most of the strains. RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at −40°C, as well as desiccation at +20°C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to −40°C. It appears that species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found in polar strains as it is also a feature of temperate strains.

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.

Protective effects of three extracts from Antarctic plants against ultraviolet radiation in several biological models

The photoprotective effect of the methanolic extracts of three Antarctic plant species - Deschampsia antarctica Desv., Colobanthus quitensis (Kunth) Bartl., and Polytrichum juniperinum Hedw. against UV-induced DNA damage was investigated in hamster lung fibroblasts (V79 cells) and in a biomonitor organism Helix aspersas, using comet assay. The protective, mutagenic, and antimutagenic profiles of these extracts were also evaluated using haploid strains of the simple eukaryote Saccharomyces cerevisiae, and antioxidant activity were investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, as well as the hypoxanthine/xanthine oxidase assay. At the concentration range employed, the extracts were not cytotoxic or mutagenic to S. cerevisiae. In addition, the treatment with these extracts enhanced survival, and decreased induced reverse, frameshift, and forward mutations in a dose-response manner in all UVC doses employed. The plants extracts did not generate DNA strand breaks in V79 cells, and the treatment significantly decreased DNA damage induced by UVC. Extracts significantly decreased UVC-induced lipid peroxidation in V79 cells, showing a clear antioxidant property. Moreover, results of comet assay in V79 cells, employing Fpg, Endo III, and Endo V enzymes, demonstrated significant reduction of UVC-induced DNA damage after pre-incubation with these extracts. The treatment with all tested extracts were much less efficient against UVC-induced cytotoxicity in the yeast strain defective in photolyase as compared to the wild type strain, suggesting that this DNA repair pathway is stimulated by substances present in the extracts. All extracts showed a significant inhibitory effect in the hypoxanthine/xanthine oxidase assay, and they had the ability to scavenge DPPH. In H. aspersas, the treatment was able to protect against UVC-induced damage. In conclusion, D. antarctica, C. quitensis, and P. juniperinum extracts present photoprotective properties, which can be attributed to molecules, such as flavonoids and carotenoids, which act as UV-absorbing molecules and as antioxidants, as well as stimulate DNA-repair processes.

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.

The family Agaricaceae Chevall. in the Tinguá Biological Reserve rain forest, Nova Iguaçú, Rio de Janeiro, Brazil: Genera Agaricus, Cystolepiota and Lepiota

Agaricaceae e a familia tipo da ordem Agaricales e apresenta especies de reconhecida importância nutricional e economica. O principal objetivo deste trabalho foi o levantamento das especies de Agaricaceae em trechos de Mata Atlântica da Reserva Biologica do Tingua, nas delimitacoes do Municipio de Nova Iguacu, Rio de Janeiro. Serao apresentadas as especies dos generos Agaricus, Cystolepiota e Lepiota coletados nas diferentes formacoes vegetais na Reserva Biologica do Tingua. Foram registrados os seguintes taxons: Agaricus subrufescens Peck., A. dulcidulus Schulzer., Lepiota lilacea Bresadola., L. tepeitensis Murril., L. forquignoni Quelet., L. echinela var rhodorhiza (Romagn. & Locq. ex. Orton) Hardtke & Rodel., L. subincarnata Lange e Cystolepiota seminuda (Lasch) Bon. Sao apresentadas chaves dicotomicas para identificacao de especies; descricoes, fotos de habito, imagens de microscopia, ilustracoes, distribuicao, habitat, dados relativos as epocas de surgimento dos basidiomas e importância das especies quando conhecida.

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.

Geospatial variability of soil CO2–C exchange in the main terrestrial ecosystems of Keller Peninsula, Maritime Antarctica

Soils and vegetation play an important role in the carbon exchange in Maritime Antarctica but little is known on the spatial variability of carbon processes in Antarctic terrestrial environments. The objective of the current study was to investigate (i) the soil development and (ii) spatial variability of ecosystem respiration (ER), net ecosystem CO2 exchange (NEE), gross primary production (GPP), soil temperature (ST) and soil moisture (SM) under four distinct vegetation types and a bare soil in Keller Peninsula, King George Island, Maritime Antarctica, as follows: site 1: moss-turf community; site 2: moss-carpet community; site 3: phanerogamic antarctic community; site 4: moss-carpet community (predominantly colonized by Sanionia uncinata); site 5: bare soil. Soils were sampled at different layers. A regular 40-point (5×8 m) grid, with a minimum separation distance of 1m, was installed at each site to quantify the spatial variability of carbon exchange, soil moisture and temperature. Vegetation characteristics showed closer relation with soil development across the studied sites. ER reached 2.26μmolCO2m(-2)s(-1) in site 3, where ST was higher (7.53°C). A greater sink effect was revealed in site 4 (net uptake of 1.54μmolCO2m(-2)s(-1)) associated with higher SM (0.32m(3)m(-3)). Spherical models were fitted to describe all experimental semivariograms. Results indicate that ST and SM are directly related to the spatial variability of CO2 exchange. Heterogeneous vegetation patches showed smaller range values. Overall, poorly drained terrestrial ecosystems act as CO2 sink. Conversely, where ER is more pronounced, they are associated with intense soil carbon mineralization. The formations of new ice-free areas, depending on the local soil drainage condition, have an important effect on CO2 exchange. With increasing ice/snow melting, and resulting widespread waterlogging, increasing CO2 sink in terrestrial ecosystems is expected for Maritime Antarctica.

Eugenia uniflora leaves essential oil induces toxicity in Drosophila melanogaster: involvement of oxidative stress mechanisms

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.

Toxicity Induced by Prasiola crispa to Fruit Fly Drosophila melanogaster and Cockroach Nauphoeta cinerea: Evidence for Bioinsecticide Action

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.