Respostas antioxidantes e dano oxidativo em diferentes regiões do corpo do poliqueto Laeonereis acuta (Polychaeta, Nereididae)

Dissertação (mestrado)-Universidade Federal do Rio Grande, Programa De Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Instituto de Ciências Biológicas, 2006.O estresse oxidativo tem o potencial de danificar macromoléculas tais como: DNA, lipídios e proteínas. O sistema de defesa antioxidante, incluindo enzimas e outros compostos de natureza não-enzimática, combate este estresse oxidativo neutralizando as espécies reativas de oxigênio. Espécies reativas de oxigênio (ERO), tais como os radicais hidroxila (OH*), são geradas através de reações químicas, sendo a reação de Haber-Weiss uma das mais importantes, produzindo este radical em presença do ânion superóxido (O2 ), peróxido de hidrogênio (H2O2) e íons ferro ou cobre. O cobre pode ser introduzido na Laguna dos Patos (RS, Brasil) pela água de drenagem oriunda de atividades de mineração no Rio Camaquã. Por outro lado, aumentos esporádicos nos níveis de cobre e de chumbo particulados podem também refletir o aporte de metais pelas atividades industriais na bacia de drenagem da Laguna dos Patos. Os poliquetos coletados na área poluída durante o verão mostraram valores de capacidade antioxidante (TOSC) para radicais peroxil similares ao longo do corpo, mas os poliquetos coletados na área não-poluída mostraram valores de TOSC maiores na região posterior em relação às regiões anterior e média e também comparado com a região posterior dos organismos coletados na área poluída. No verão, a concentração de ERO foi maior nas regiões anterior e média nos poliquetos da área poluída em relação aos poliquetos da área não poluída. Durante o inverno as três regiões dos organismos da área poluída mostraram maiores concentrações de ERO. No experimento laboratorial, não houve diferença na atividade da catalase nas distintas regiões do corpo do poliqueto exposto ao cobre. A atividade da glutationa S-transferase na região anterior foi maior no poliqueto exposto ao cobre. Os valores de TOSC foram similares para radicais hidroxila no grupo exposto e no grupo controle. Já para os radicais peroxil houve uma queda no grupo exposto ao cobre em relação ao grupo controle. No entanto, os valores de TOSC para radicais peroxil sempre foram maiores que os de radicais hidroxilas. A lipoperoxidação foi menor na região posterior em ambos tratamentos. O dano de DNA avaliado pelo método cometa foi maior na região anterior exposta ao cobre. Os poliquetas do grupo exposto acumularam cobre igualmente em todas regiões

Variation in oxygen disponibility and antioxidants responses in the gastropod Helix aspersa

O gastrópode terrestre Helix aspersa (Müller) é um herbívoro generalista, que habita a região mediterrânea. Os gastrópodes terrestres em geral entram em estados dormentes durante o seu ciclo de vida. A dormência é uma forma de inatividade associada a uma redução na taxa metabólica, sem grandes alterações no estado hídrico do animal (Withers & Cooper, 2010). Os gastrópodes terrestres quando saem de um estado dormente podem apresentar um aumento na produção de espécies reativas de oxigênio (ROS) nas mitocôndrias (Turrens et al., 1982) levando a um quadro de possível estresse oxidativo (Hermes-Lima & Zenteno-Savin, 2002). Cerca de 0,1% a 2% da respiração normal celular in vitro resulta em formação de ânion superóxido (Fridovich, 2004; Murphy, 2009; Hamanaka & Chandel, 2010). Muitos estudos apontam para um aumento na produção de ROS (Duranteau et al., 1998; Chandel et al., 1998; Wood et al., 1999; Killilea et al., 2000) durante a hipóxia. O estresse oxidativo é definido como o desequilíbrio no balanço entre agentes pró-oxidantes e agentes antioxidantes, em favor dos pró-oxidantes, levando a uma perturbação na sinalização e no controle redox e/ou dano molecular (Sies & Jones, 2007). A GSH é o principal grupo sulfidrila não proteico encontrado em células de mamíferos. Esta normalmente em uma concentração de 1 a 10 mM, enquanto a GSSG é encontrada em uma concentração de 10 a 100 vezes menor (Rossi et al., 1995; Griffith, 1999). A GSH atua desativando radicais livres, preservando o status redox celular e defendendo o organismo contra xenobióticos (Meister, 1995a). A ativação do sistema de defesa antioxidante, incluindo aumento da atividade de enzimas antioxidantes, durante situações de depressão metabólica foi chamada de preparo para o estresse oxidativo (Hermes-Lima et al., 1998). Esta ativação protege o organismo durante o hipometabolismo e durante a reoxigenação/despertar de um possível estresse oxidativo. Os objetivos deste estudo foram: analisar as possíveis respostas durante um ciclo de anoxia e reoxigenação do sistema de defesa antioxidante de Helix aspersa com níveis reduzidos de glutationa total (eq-GSH); e examinar a liberação de ROS em mitocôndrias isoladas de Helix aspersa em estivação. O metabolismo de GSH mostrou-se em nosso estudo como importante fator na manutenção do equilíbrio redox de Helix aspersa durante a anoxia e reoxigenação, lidando com um provável aumento de produção de ROS durante a reoxigenação. E durante a estivação, foi demonstrado que as mitocôndrias de glândula digestiva de Helix aspersa liberam mais H2O2 in vitro. Este aumento na liberação de ROS na mitocôndria pode estar relacionado com a indução de respostas antioxidantes, que ocorrem durante a estivação em gastrópodes terrestres em diversos estudos (Hermes-Lima & Storey, 1995; Ramos-Vasconcelos & Hermes-Lima, 2003; Ramos-Vasconcelos et al., 2005)The gastropod Helix aspersa (Müller) is a generalist herbivore that inhabits the Mediterranean region. The terrestrial gastropods generally go into dormant states during their life cycle. Dormancy is a form of inactivity associated with a reduction in metabolic rate, without major changes in the water status of the animal (Withers & Cooper, 2010). The terrestrial gastropods when they leave a dormant state may experience an increased production of reactive oxygen species (ROS) in mitochondria (Turrens et al., 1982) leading to a potential oxidative stress (Hermes-Lima & Zenteno-Savin, 2002). About 0.1% to 2% of the normal cellular respiration in vitro results in formation of superoxide anion (Fridovich, 2004; Murphy, 2009; Hamanaka & Chandel, 2010). Many studies point to an increased production of ROS (Duranteau et al. 1998; Chandel et al., 1998, Wood et al. 1999; Killilea et al., 2000) during hypoxia. Oxidative stress is defined as the imbalance between pro-oxidant agents and antioxidants in favor of pro-oxidants, leading to a disruption of redox signaling and redox control and/or molecular damages (Sies & Jones, 2007). GSH is the main non-protein sulfhydryl group found in mammalian cells. It´s usually in a concentration of 1 to 10 mM, whereas GSSG is found at a concentration of 10 to 100 times lower (Rossi et al. 1995; Griffith, 1999). GSH acts by disabling free radicals, maintaining the cellular redox status and defending the body against xenobiotics (Meister, 1995a). The activation of the antioxidant defense system, including increased activity of antioxidant enzymes, during situations of metabolic depression is called \"preparation for oxidative stress (Hermes-Lima et al., 1998). This activation protects the body during hypometabolism and during recovery of a possible situation of oxidative stress. The objectives of this study were: to analyze the possible response during a cycle of anoxia and reoxygenation of the antioxidant defense system of Helix aspersa with reduced levels of total glutathione (GSH-eq) and to examine the release of ROS in isolated mitochondria from Helix aspersa in aestivation. The metabolism of GSH presented itself in our study as an important factor in maintaining the redox balance of Helix aspersa during anoxia and reoxygenation, dealing with a probable increase in ROS production during reoxygenation. And during aestivation, it was demonstrated that the digestive gland mitochondria of Helix aspersa released more H2O2 in vitro. This increased release of ROS in mitochondria may be related to induction of antioxidant responses that occur during aestivation in terrestrial gastropods in several studies (Hermes-Lima & Storey, 1995; Ramos-Vasconcelos & Hermes-Lima, 2003, Ramos- Vasconcelos et al., 2005

Glutathione Depletion Disrupts Redox Homeostasis in an Anoxia-Tolerant Invertebrate

The upregulation of endogenous antioxidants is a widespread phenomenon in animals that tolerate hypoxia/anoxia for extended periods. The identity of the mobilized antioxidant is often context-dependent and differs among species, tissues, and stresses. Thus, the contribution of individual antioxidants to the adaptation to oxygen deprivation remains elusive. This study investigated the role of glutathione (GSH) in the control of redox homeostasis under the stress of anoxia and reoxygenation in Helix aspersa, an animal model of anoxia tolerance. To do so, the total GSH (tGSH) pool was depleted with l-buthionine-(S, R)-sulfoximine (BSO) before exposing snails to anoxia for 6 h. Then, the concentration of GSH, glutathione disulfide (GSSG), and oxidative stress markers (TBARS and protein carbonyl) and the activity of antioxidant enzymes (catalase, glutathione peroxidase, glutathione transferase, glutathione reductase, and glucose 6-phosphate dehydrogenase) were measured in foot muscle and hepatopancreas. BSO alone induced tGSH depletion by 59–75%, but no other changes happened in other variables, except for foot GSSG. Anoxia elicited a 110–114% increase in glutathione peroxidase in the foot; no other changes occurred during anoxia. However, GSH depletion before anoxia increased the GSSG/tGSH ratio by 84–90% in both tissues, which returned to baseline levels during reoxygenation. Our findings indicate that glutathione is required to withstand the oxidative challenge induced by hypoxia and reoxygenation in land snails

Antioxidant responses in different body regions of the polychaeta Laeonereis acuta (Nereididae) exposed to copper

Antioxidant enzymes, total antioxidant capacity (TOSC) and concentration of reactive oxygen species (ROS) were measured in anterior (A), middle (M) and posterior (P) body regions of Laeonereis acuta after copper (Cu; 62.5 mg/l) exposure. A catalase (CAT) activity gradient observed in control group (lowest in A, highest in P) was not observed in Cu exposed group. Glutathione-S-transferase (GST) activity in A region of Cu group was higher than in A region of the control group. DNA damage (comet assay) was augmented in the A region of Cu group. Since copper accumulation was similar in the different body regions, sensitivity to copper in A regions seems to be related to lowest CAT activity. In sum, copper exposure lowered TOSC, a result that at least in part can be related to lowering of antioxidant enzymes like CAT. DNA damage was induced in the anterior region, where a lower CAT activity was observed

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

Pollution biomarkers in estuarine animals: Critical review and new perspectives

In this review, recent developments in monitoring toxicological responses in estuarine animals are analyzed, considering the biomarker responses to different classes of pollutants. The estuarine environment imposes stressful conditions to the organisms that inhabit it, and this situation can alter their sensitivity to many pollutants. The specificity of some biomarkers like metallothionein tissue concentration is discussed in virtue of its dependence on salinity, which is highly variable in estuaries. Examples of cholinesterase activity measurements are also provided and criteria to select sensitive enzymes to detect pesticides and toxins are discussed. Regarding non-specific biomarkers, toxic responses in terms of antioxidant defenses and/or oxidative damage are also considered in this review, focusing on invertebrate species. In addition, the presence of an antioxidant gradient along the body of the estuarine polychaete Laeonereis acuta (Nereididae) and its relationship to different strategies, which deal with the generation of oxidative stress, is reviewed. Also, unusual antioxidant defenses against environmental pro-oxidants are discussed, including the mucus secreted by L. acuta. Disruption of osmoregulation by pollutants is of paramount importance in several estuarine species. In some cases such as in the estuarine crab Chasmagnathus granulatus, there is a trade off between bioavailability of toxicants (e.g. metals) and their interaction with key enzymes such as Na+–K+-ATPase and carbonic anhydrase. Thus, the metal effect on osmoregulation is also discussed in the present review. Finally, field case studies with fish species like the croaker Micropogonias furnieri (Scianidae) are used to illustrate the application of DNA damage and immunosuppressive responses as potential biomarkers of complex mixture of pollutants

Toxicological responses in Laeonereis acuta (Annelida, Polychaeta) after arsenic exposure

Several environmental pollutants, including metals, can induce oxidative stress. So, the objective of this study was to evaluate the effects of arsenic (AsIII, as As2O3) on the antioxidant responses in the polychaete Laeonereis acuta. Worms were exposed to two environmentally relevant concentrations of As, including the highest previously allowed by Brazilian legislation (50 μg As/l). A control group was kept in saline water(10‰) without added metal. It was observed that: (1) a peak concentration of lipid peroxide was registered after 2 days of exposure to 50 μg As/l (61±3.2 nmol CHP/g wet weight)compared to the control group (43±4.5 nmol CHP/g wet weight), together with a lowering of the activity of the antioxidant enzyme catalase (−47 and −48%, at 50 or 500 μg As/l respectively) and a higher superoxide dismutase activity (+305% at 50 μg As/l with respect to the control group); (2) a lower conjugation capacity through glutathione-S-transferase activity was observed after 7 days of exposure to 50 μg As/l (−48% compared to the control group); (3) a significant increase in As concentration was verified after 1 week of exposure to both As concentrations 50 and 500 μg/l); (4) worms exposed to As showed a limited accumulation of related methylated As species and the levels of non-toxic As species like arsenobetaine (AsB) and arsenocholine (AsC) remained unchanged during the exposure period when compared with the controls. Overall, it can be concluded that As interfered in the antioxidant defense system of L. acuta, even at low concentrations (50 μg/l)that Brazilian legislation previously considered safe. The fact that worms exposed to As showed high levels of methylated As species indicates the methylation capability of L. acuta, although the high levels of inorganic As suggest that not all the administered AsIII (as As2O3) is completely removed or biotransformed after 7 days of exposure