Influência da exposição in vitro ao fulereno c60 no estado redox e peroxidação lipídica de cérebro e brânquias da carpa Cyprinus carpio (Teleostei, Cyprinidae)

Dissertação (mestrado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Instituto de Ciências Biológicas, 2009.Os estudos sobre o impacto das nanopartículas, especialmente o fulereno C60 em ambientes de água doce e seus efeitos sobre a fisiologia dos organismos aquáticos ainda permanecem em pequeno número e com resultados conflitantes. Isso porque nanocompostos em geral, incluindo os derivados do carbono, estão sujeitos a inúmeras variáveis que podem levar a modificações estruturais, elétricas e químicas durante a exposição in vivo de um organismo, o que afeta diretamente sua toxicidade, a qual acredita-se estar vinculada à produção de espécies reativas de oxigênio (ERO). Levando em consideração um conceito emergente de estresse oxidativo, no qual as ERO atuam como moléculas sinalizadoras e de controle de processos redox celulares, o presente estudo teve como objetivo verificar o efeito in vitro do fulereno C60 em extratos de cérebro e brânquias de carpa (Cyprinus carpio) durante 1, 2 e 4 horas de exposição, avaliando os seguintes parâmetros de estado redox: níveis de peroxidação lipídica, estado de oxidação do tripeptídeo glutationa (GSH/GSSG) e do aminoácido cisteína (Cys/CySSyC), capacidade antioxidante total e atividade das enzimas glutationa redutase (GR) e glutationa-S-transferase (GST). O fulereno induziu aumento significativo (p<0.05) em lipídios peroxidados após 2 hs em ambos os órgãos, inibiu a atividade da GR em brânquias (1 h) e cérebro (4 hs) e reduziu a capacidade antioxidante no cérebro (4 hs), aumentando os níveis de GSSG no cérebro em 1 h e reduzindo-os em 2 hs. Considerando esses resultados, pode-se inferir que o fulereno pode interferir no estado redox celular através de mecanismos tiol/dissulfeto, levando ao dano oxidativo e perda de competência antioxidante. Esse tipo de distúrbio possui implicações importantes, pois mecanismos de controle redox envolvendo grupos tiol fazem parte de vários processos celulares que, até o momento, não estão sendo estudados pela nanotoxicologia

Antioxidant responses in the polychate Perinereis gualpensis (Nereididae) after exposure to the carbon nanomaterial fullerene (C60) in sediments

The objective of this study was to analyse biochemical responses induced by the carbon nanomaterial fullerene (C60) in the polychaete Perinereis gualpensis(Nereididae). The activity of glutathione-Stransferase (GST), glutathione reductase (GR) and glutamate cysteine ligase (GCL), as well as total antioxidant capacity, concentration of glutathione (GSH) and malondialdehyde (TBARS), were analysed. Estuarine worms were maintained in sediments collected at an unpolluted site and spiked with fullerene (3 mg C60·g−1 sediment).Acontrol groupwas run in parallel. Scanning electron microscope (SEM) images of sediment and fullerene indicated that the size of the carbon nanomaterial should enable it to be ingested by the polychaete. No evidence of oxidative damage (TBARS) was observed in any of the treatments, and the same was true for GSH and GCL measurements (p > 0.05). Total antioxidant capacity was higher in the C60 group after 2 and 7 d when compared with the control group (p < 0.05), suggesting that fullerene is acting as an antioxidant. The fact that P. gualpensis is an infaunal organism diminishes the chance of fullerene photoexcitation with consequent reactive oxygen species production. Thus, the data indicated an absence of toxic responses mediated by oxidative stress in estuarine worms exposed to C60 mixed in sediments

A method to measure total antioxidant capacity against peroxyl radicals in aquatic organisms: application to evaluate microcystins toxicity

Determination of total antioxidant capacity, instead of the measurements of limited number of antioxidants, is very important for the understanding of how antioxidants interact with reactive oxygen species (ROS). Several techniques already exist with this propose, although some of them are extremely time-consuming. A new methodology is proposed, based on the detection of ROS by fluorometry (ex/em: 485/520 nm) employing 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) as substrate. Supernatant of homogenized samples from different organs (gill, muscle, liver, and brain) of the teleost fish Jenynsia multidentata (Anaplebidae) were exposed to peroxyl radicals generated by thermal (35 °C)decomposition of 2,2′-azobis (2 methylpropionamidine) dihydrochloride (ABAP, 4 mM). Different protein concentrations (0.5, 1, 2 and 8 mg/ml) were assayed to get the best signal and curve fitting of fluorescence data over time (30 min). Total antioxidant capacity against peroxyl radicals was estimated as the difference in ROS area with and without ABAP, relative to the fluorescence registered without ABAP. For application of this methodology,J. multidentata specimens were exposed for 24 h to microcystins, cyanotoxins known to induce oxidative stress. Almost all organs showed a lower antioxidant capacity (pb0.05) in samples with 8 mg proteins/ml, when compared to protein content of 1–2 mg/ml. In liver samples, higher (pb0.05) free iron content was determined in samples with 8mg proteins/ml.Sensitivity test employing GSH spiked in homogenized samples showed the protocol efficiency in detecting total antioxidant capacity. In the test with microcystins a dosedependent decrease (pb0.05) of antioxidant competence in gills and brain and an inverse result with liver samples were observed. The use of antioxidant defenses was efficient in avoiding oxidative damage, as the content of oxidized proteins was not altered. Data obtained show the potential of this new methodology to be used in ecotoxicological studies

Antioxidant responses and reactive oxygen species generation in different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae)

The aim of this study was to analyze the total antioxidant capacity (TOSC), generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) in the different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae) sampled at non-polluted (NOPOL)and polluted (POL) sites from Lagoa dos Patos (Southern Brazil). Organisms collected at POL during summer showed similar (p > 0.05)TOSC values along the body, but worms collected at NOPOL presented higher (p < 0.05) TOSC values in the posterior (P) region in respect of anterior (A) region and middle (M) region. TOSC in the P region at NOPOL was higher (p < 0.05) compared with the same body region of worms at POL. In summer, ROS concentration was higher in A andMregions of worms at POL in respect of the organisms at NOPOL. During winter all the regions showed higher ROS in worms sampled at POL. It was registered absence of season influence on LPO content,but in the P region at NOPOL in summer there were lower LPO levels compared with the others regions (p < 0.05). In vitro assays showed that P region, despite a higher basal ROS, presented a higher competence to cope with pro-oxidants compared with A and M regions (p < 0.05), corroborating the field results. A lower proteic sulfhydril content was observed in P in respect of the other regions (p < 0.05) supporting the idea of a highest oxidant condition in this region. The results indicate that worms collected at the POL site are confronted to higher ROS concentrations, affecting its antioxidant capacity, a result that depends of body regions

The benefits and drawbacks of nicotine exposure in the cortex and hippocampus of old rats

Nicotine is the main alkaloid of tobacco and possesses well-established stimulant effects. Previous reports show that nicotine at low doses improves memory functions, while high doses impair memory. This study aims to analyze the effects of nicotine (NIC) on inhibitory avoidance task and on DNA damage, reactive oxygen species (ROS) concentration, total antioxidant capacity, and lipid peroxidation in cortex and hippocampus of old rats. Male Wistar rats of 24–26 months old (620–700 g) were exposed i.p. to two doses (0.3 and 1 mg/kg) of NIC daily during 9 days. The treatment NIC 0.3 enhanced long-term memory ( p < 0.05), whereas NIC 1 improved both short and long-term memories ( p < 0.05). DNA damage was observed only in hippocampus ( p < 0.05) after NIC 1 exposure. A similar result was obtained for ROS: higher levels were detected at NIC 1 treatment in hippocampus ( p < 0.05). No alterations in the total antioxidant capacity were verified after NIC exposure (0.3 and 1 mg/kg) in both tissues ( p > 0.05). Finally, evidence of oxidative damage was observed in terms of lipid peroxides levels, being higher at NIC 1 in hippocampus ( p < 0.05). Overall the results indicate that deleterious effects paralleled the improved short and long-term memories at the highest NIC dose, since augmented DNA damage, ROS concentration and lipid peroxides levels were registered

Modulation of antioxidant and detoxification responses mediated by lipoic acid in the fish Corydoras paleatus (Callychthyidae)

Lipoic acid (LA) has been reported as a potential therapeutic agent due its antioxidants proprieties. It was considered its effect in different organs (gills, brain, muscle and liver) of the fish Corydoras paleatus(Callychthyidae). LA (70 mg/kg of body mass) was added to a commercial fish diet, organisms being fed daily (1% body weight). Sixty animals (mean mass: 2.37±0.09 g) were placed randomly in aquariums and received (+LA) or not (−LA) lipoic acid enriched diet during four weeks. After, fish were killed and the brain, muscle, gills and liver were dissected. LA treatment reduced significantly (pb0.05) reactive oxygen species concentration in brain and increased (pb0.05) glutamate-cysteine ligase activity in brain and liver of the same experimental group. LA fed organisms showed higher (pb0.05) brain glutathione-S-transferase activity, indicating that LA improves the detoxification and antioxidant capacity face components that waste glutathione in phase II reactions. A conspicuous reduction of protein oxidationwas observed inmuscle and liver of +LA organisms, indicating that the treatment was also effective in reducing oxidative stress parameters

Biomonitoring of antioxidant and oxidative stress responses in Perinereis gualpensis (Polychaeta: Nereididae) in Chilean estuarine regions under different anthropogenic pressure

This study aimed to analyze oxidative stress parameters including levels of the antioxidant glutathione (GSH),activity of glutamate-cysteine ligase (GCL) and glutathione-S-transferase (GST), total antioxidant capacity and protein oxidation, in the polychaete Perinereis gualpensis (Nereididae) collected from the Biobı´o, Itata, Valdivia and Lingue estuaries in Chile,which present different degrees of anthropogenic pressure. Sampling sites were characterized considering a geographic information system and the physicochemical characteristics of water and sediment. Significant differences (po0.05) were observed between the sampling sites for most of the responses (GSH, GCL, GST and antioxidant capacity), mainly related to human activities such as agriculture, industry, amongothers. Multivariate correlation analysis indicates acertain relationship of antioxidant responses with human activities, salinity, and worm weight, this last employed to standardize GST and antioxidant capacity. These results clearly indicate biomarker responses in P. gualpensis in Biobı´o and Valdivia estuaries, the more affected by human activities

In vitro exposure to fullerene c60 influences redox state and lipid peroxidation in brain and gills from cyprinus carpio (cyprinidae)

Studies concerning the impact of nanomaterials, especially fullerene (C60), in fresh water environments and their effects on the physiology of aquatic organisms are still scarce and conflicting. We aimed to assess in vitro effects of fullerene in brain and gill homogenates of carp Cyprinus carpio, evaluating redox parameters. A fullerene suspension was prepared by continued stirring under fluorescent light during two months. The suspension concentration was measured by total carbon content and ultraviolet–visible spectroscopy nephelometry. Characterization of C60 aggregates was performed with an enhanced dark-field microscopy system and transmission electronic microscopy. Organ homogenates were exposed during 1, 2, and 4 h under fluorescent light. Redox parameters evaluated were reduced glutathione and oxidized glutathione, cysteine and cystine, total antioxidant capacity; activity of the antioxidant enzymes glutathione S-transferase and glutathione reductase (GR), and lipid peroxidation (TBARS assay). Fullerene induced a significant increase ( p<0.05) in lipid peroxidation after 2 h in both organs and reduced GR activity after 1 h (gills) and 4 h (brain) and antioxidant capacity after 4 h (brain). Levels of oxidized glutathione increased in the brain at 1 h and decreased at 2 h as well. Given these results, it can be concluded that C60 can induce redox disruption via thiol/disulfide pathway, leading to oxidative damage (higher TBARS values) and loss of antioxidant competence

Antioxidant properties of the mucus secreted by Laeonereis acuta (Polychaeta, Nereididae): a defense against environmental pro-oxidants?

Polychaeta species like Laeonereis acuta (Nereididae) usually secrete great amounts of mucus that wrap the animal inside. Taking into account that fungi action in the sediment and UV radiation acting on dissolved organic matter in the water produces reactive oxygen species (ROS) like hydrogen peroxide (H2O2), it was considered that the mucus secretion could represent an antioxidant defense against environmental ROS. Antioxidant enzymes (catalase—CAT; superoxide dismutase—SOD; glutathione peroxidase—GPx and glutathione-S-transferase—GST) and total antioxidant capacity (TOSC) were determined in worms and mucus secretion. Higher (pb0.05) CAT, GPx and TOSC values were registered in mucus samples respect worms, SOD activity was similar (pN0.05) in both kind of samples, and absence of GST activity was observed in mucus samples, suggesting absence of catalyzed phase II reactions. In assays conducted with hepatoma cell lines exposed to H2O2, it was verified that:(1) mucus co-exposure significantly (pb0.05) lowered DNA damage induced by H2O2; (2) ROS production was significantly (pb0.05) reduced when cells were exposed simultaneously with mucus samples and H2O2 respect H2O2 alone. It can be concluded that the mucus production contributes substantially to the antioxidant defense system of the worm against environmental ROS through the interception or degradation of H2O2, peroxyl and hydroxyl radicals