Rafael Trevisan

Involvement of glutathione, ERK1/2 phosphorylation and BDNF expression in the antidepressant-like effect of zinc in rats

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.

A light in the darkness: new biotransformation genes, antioxidant parameters and tissue-specific responses in oysters exposed to phenanthrene.

Phenanthrene (PHE), a major component of crude oil, is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystems, and is readily bioavailable to marine organisms. Understanding the toxicity of PAHs in animals requires knowledge of the systems for xenobiotic biotransformation and antioxidant defence and these are poorly understood in bivalves. We report, for the first time, new transcripts and tissue-specific transcription in gill and digestive gland from the oyster Crassostrea brasiliana following 24h exposure to 100 and 1000μgL(-1) PHE, a model PAH. Six new cytochrome P450 (CYP) and four new glutathione S-transferase (GST) genes were analysed by means of quantitative reverse transcription PCR (qRT-PCR). Different antioxidant endpoints, including both enzymatic and non-enzymatic parameters, were assessed as potential biomarkers of oxidative stress. GST activity was measured as an indicator of phase II biotransformation. Rapid clearance of PHE was associated with upregulation of both phase I and II genes, with more pronounced effects in the gill at 1000μgL(-1) PHE. After 24h of exposure, PHE also caused impairment of the antioxidant system, decreasing non-protein thiols and glutathione levels. On the other hand, no change in antioxidant enzymes was observed. PHE treatment (100μgL(-1)) significantly decreased GST activity in the gill of exposed oysters. Both CYP and GST were transcribed in a tissue-specific manner, reflecting the importance of the gill in the detoxification of PAHs. Likewise, the antioxidant parameters followed a similar pattern. The data provide strong evidence that these genes play key roles in C. brasiliana biotransformation of PHE and highlight the importance of gill in xenobiotic metabolism.

Cellular and Transcriptional Responses of Crassostrea gigas Hemocytes Exposed in Vitro to Brevetoxin (PbTx-2)

Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes.

Selenium in water enhances antioxidant defenses and protects against copper-induced DNA damage in the blue mussel Mytilus edulis.

Selenium and copper are naturally occurring elements in the environment that have important roles in cellular function. Selenium is known for its role in antioxidant defense, whereas copper is a redox-active metal capable of acting as a pro-oxidant. We investigated the effects of short term selenium (Na(2)SeO(3)) supplementation (4 μg/L for 3 days) on antioxidant parameters of the blue mussel, Mytilus edulis, and its possible protective effects against a subsequent copper (CuSO(4)) exposure (56 μg/L for 3 days). Selenium supplementation caused a 4-fold increase in glutathione levels in gills. The activity of selenium-dependent glutathione peroxidase was modulated by selenium in gills (2-fold increase) and also in cell-free haemolymph (40% increase). Copper exposure produced decreases in protein thiol levels (35%) and in thioredoxin reductase activity (60%) in gills and induced an increase in DNA damage in haemocytes (70% increase in % tail DNA observed using the comet assay). The decrease in thioredoxin reductase activity may constitute a mechanism of copper toxicity in bivalves, warranting further investigation. Pre-treatment with selenium largely prevented these deleterious effects of copper on protein thiols, thioredoxin reductase activity and DNA damage. The results suggest that induction of key antioxidant defenses such as glutathione and selenium-dependent glutathione peroxidase, as a result of selenium supplementation, may play an important role in protection of aquatic organisms against oxidative stress.

Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 μm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC50 (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte count). At 24 h post exposure, decreased (-29%) glutathione reductase (GR) activity was observed in gills, but other biochemical responses were observed only after 48 h of exposure: lower GR activity (-28%) and levels of protein thiols (-21%), increased index of lipid peroxidation (+49%) and GPx activity (+26%). In accordance with ultrastructural changes and zinc load, digestive gland showed delayed biochemical responses. Except for a decreased GR activity (-47%) at 48 h post exposure, the biochemical alterations seen in gills were not present in digestive gland. The results indicate that gills are able to incorporate zinc prior (24 h) to digestive gland (48 h), leading to earlier mitochondrial disruption and oxidative stress. Our data suggest that gills are the initial target of ZnONP and that mitochondria are organelles particularly susceptible to ZnONP in C. gigas.

Antioxidant and Acetylcholinesterase Response to Repeated Malathion Exposure in Rat Cerebral Cortex and Hippocampus

The wide use of the organophosphate insecticide malathion is accompanied by the risk of human exposure, especially in developing countries, which underlines the need of basic studies in this area. Some reports have shown that low doses of malathion, in a repeated treatment regimen, are unable to reduce acetylcholinesterase (AChE) activity in the rat brain, in contrast to the inhibitory effect in acute treatment. In order to investigate if AChE activity is affected by repeated low-level malathion administration, female Wistar rats were exposed to malathion (50 and 100 mg/kg, intraperitoneally) for 3 consecutive days. Exposure to malathion 50 mg/kg did not affect AChE activity, as previously observed. Contrary to expectation, 100 mg/kg malathion produced a significant increase in AChE activity in both cerebral cortex and hippocampus. Besides AChE inhibition, malathion may act as a pro-oxidative agent by interfering with antioxidant defences, as shown by a decrease of glutathione peroxidase and glutathione reductase activity in the cerebral cortex (100 mg/kg malathion). These effects are in contrast to response in the hippocampus where the increase in AChE activity correlates positively with the antioxidant defences, while the opposite was found in the cerebral cortex. These data indicate that, with low doses, and after a short period of exposure, malathion induces an up-regulation of AChE activity, a pattern similar to that found in the hippocampus for the antioxidant defences studied. The cerebral cortex was more vulnerable to malathion, as reflected in a decrease of two antioxidant enzymes. This study indicates that (i) alternatively to AChE inhibition, interference with the antioxidant defence system may be another important target for malathion toxicity; (ii) hippocampal and cortical AChE activity in rats can be increased after repeated low-dose malathion exposure. This response suggests the occurrence of a pathophysiological response in order to maintain the homeostasis of the cholinergic system in these cerebral structures.

Folic acid administration prevents ouabain-induced hyperlocomotion and alterations in oxidative stress markers in the rat brain.

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 microM), 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 microM, 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.

Phylogenetic Relationships in Eleocharis (Cyperaceae): C4 Photosynthesis Origins and Patterns of Diversification in the Spikerushes

Abstract Eleocharis (Cyperaceae) is a morphologically and physiologically diverse lineage of 250 + species with a cosmopolitan distribution. We here explore phylogenetic relationships in this lineage using maximum parsimony and Bayesian inference analyses of nrDNA ITS and cpDNA tmC-ycf6 and ycf6-psbM sequence data with the goals of comparing our phylogenetic hypotheses to previous classifications, morphological variation, and photosynthetic pathway variation. Our results suggest that in Eleocharis C4 photosynthesis has been derived at least three times, with several cases of possible reversion to C3-like or intermediate pathways and several additional origins of C3–C4 intermediate photosynthetic pathways, as inferred by carbon isotope ratio measurements. Many classification units currently recognized in Eleocharis are not monophyletic, however, E. subgenus Limnochloa and E. subgenus Scirpidium are monophyletic. Other classification units largely corresponding to clades include E. subgenus Zinserlingia, E. subseries Chaetariae, and E. series Maculosae. Problems with species circumscription and morphological variation in several groups are discussed in light of the phylogeny, particularly in the context of species membership of seven focal clades found in the analyses.

Zinc causes acute impairment of glutathione metabolism followed by coordinated antioxidant defenses amplification in gills of brown mussels Perna perna

Zinc demonstrates protective and antioxidant properties at physiological levels, although these characteristics are not attributed at moderate or high concentrations. Zinc toxicity has been related to a number of factors, including interference with antioxidant defenses. In particular, the inhibition of glutathione reductase (GR) has been suggested as a possible mechanism for acute zinc toxicity in bivalves. The present work investigates the biochemical effects of a non-lethal zinc concentration on antioxidant-related parameters in gills of brown mussels Perna perna exposed for 21 days to 2.6 μM zinc chloride. After 2 days of exposure, zinc caused impairment of the antioxidant system, decreasing GR activity and glutathione levels. An increase in antioxidant defenses became evident at 7 and 21 days of exposure, as an increase in superoxide dismutase and glutathione peroxidase activity along with restoration of glutathione levels and GR activity. After 7 and 21 days, an increase in cellular peroxides and lipid peroxidation end products were also detected, which are indicative of oxidative damage. Changes in GR activity contrasts with protein immunoblotting data, suggesting that zinc produces a long lasting inhibition of GR. Contrary to the general trend in antioxidants, levels of peroxiredoxin 6 decreased after 21 days of exposure. The data presented here support the hypothesis that zinc can impair thiol homeostasis, causes an increase in lipid peroxidation and inhibits GR, imposing a pro-oxidant status, which seems to trigger homeostatic mechanisms leading to a subsequent increase on antioxidant-related defenses.

Biochemical alterations in juvenile carp (Cyprinus carpio) exposed to zinc: glutathione reductase as a target.

The aim of this study was to investigate biochemical changes in juvenile carp (Cyprinus carpio) exposed to zinc chloride (10, 30 and 100 microM) for a period of 48 h. Zinc exposure caused a concentration-dependent reduction in glutathione reductase (GR) activity in gills, liver and brain. Gill glutathione S-transferase (GST) was reduced when animals were exposed to the highest concentration of 100 microM zinc. The phosphorylation of p38(MAPK) increased in the brain of fish exposed to zinc 100 microM, while phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1/2 (JNK1/2) remained unchanged. Expression of proteins HSP60 and HSP70 were not affected by zinc exposure. Considering the significant concentration-dependent inhibition of GR in all tissues analyzed, this enzyme could be a potential biomarker of exposure to zinc, which has to be confirmed.