Oxygen radical formation in anoxic transgression and anoxia-reoxygenation: foe or phantom? Experiments with a hypoxia tolerant bivalve.

Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. We exposed mussels to experimental short and prolonged anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on reactive oxygen species (mainly ROS: superoxide anion, O2·- and hydrogen peroxide, H2O2), formation using live imaging techniques and confocal microscopy. Our aim was to understand if this "natural stress" would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO2 in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO2 range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical pc values (pc1: onset of oxyregulation in gills, pc2: switch from oxyregulation to oxyconformity) to higher pO2. Concentrations of both ROS decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first paper that directly measures ROS formation during anoxia reoxygenation in mussels. We conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions.

Chromium induced stress conditions in heterotrophic and auxotrophic strains of Euglena gracilis.

Oxidative stress parameter and antioxidant defense compound as well as enzyme activity were studied in relation to different Cr(VI) concentrations (0, 10, 20, 40 µM) in two strains of Euglena gracilis, one isolated from a polluted river (MAT) and the other acquired from a culture collection (UTEX). Chromium toxicity was measured in the auxotrophic and obligated heterotrophic variants of the two strains. Chromium uptake was higher in auxotrophic cultures, reflected by their higher cell proliferation inhibition and lower IC50 levels compared to heterotrophic ones. In the Cr(VI) treatments a reduction of chlorophyll a and b ratio (Chl a/Chl b) was observed, the ratio of protein to paramylon content was augmented, and total lipid content increased, having the auxotrophic strains the highest values. TBARS content increased significantly only at 40 µM Cr(VI) treatment. Unsaturated fatty acids also increased in the Cr(VI) treatments, with the higher storage lipid (saturated acids) content in the heterotrophic cells. The antioxidant response, such as SOD activity and GSH content, increased with chromium concentration, showing the highest GSH values in the heterotrophic cultures and the SOD enzyme participation in chromium toxicity. The MAT strain had higher IC50 values, higher carbohydrate and saturated acid content, and better response of the antioxidant system than the UTEX one. This strain isolated from the polluted place also showed higher GSH content and SOD activity in control cells and in almost all treated cultures. SOD activity reached a 9-fold increase in both MAT strains. These results suggest that tolerance of MAT strain against Cr(VI) stress is not only related to GSH level and/or biosynthesis capacity but is also related to the participation of the SOD antioxidant enzyme.

Oxidative stress and histological alterations produced by dietary copper in the fresh water bivalve Diplodon chilensis.

The aim of this work was to study the oxidative stress effects and histological alterations caused by dietary copper on the filter-feeding freshwater mussel Diplodon chilensis. Bivalves were fed during 6 weeks with the green algae Scenedesmus vacuolatus previously exposed to copper. Metal concentration in algae cultures and bivalve digestive gland was measured by TXRF. A maximum accumulation of 0.49 µg Cu/mg protein was detected at week 6. Also at this week, the hepatosomatic index (HSI) showed the highest decrease (50%) in response to Cu exposure. SOD and GST activities were significantly increased at weeks 4, 5 and 6, reaching an activity on average 50% higher than in controls for GST. CAT activity and GSH increased significantly at weeks 5 and 6. Despite this response, oxidative damage measured as TBARS and carbonyl groups contents increased significantly at weeks 4, 5 and 6, respectively. Digestive tubule and duct atrophy and cell-type replacement in treated mussels were observed by histological studies. The presence of intracellular rhodanine-positive granules, suggests copper accumulation in intracellular vacuoles of digestive cells.

Effect of chromium on the fatty acid composition of two strains of Euglena gracilis.

The effect of hexavalent chromium on fatty acid composition was studied in two strains of Euglena gracilis; UTEX 753 (from the Culture Collection of Algae of Texas University, USA) and MAT (isolated from a highly polluted River). Both were grown in photoauxotrophic and photoheterotrophic conditions and exposed to two metal concentrations, one below and one above IC50. The high malondialdehyde (MDA) levels (3 to 7-fold) obtained with chromium concentration above IC50, suggested the existence of metal-induced lipid peroxidation. Total lipid content increased only with concentration below IC50, whereas it was inhibited by higher metal concentration. Photoheterotrophic control strains exhibited a significantly higher proportion of saturated and polyunsaturated fatty acids. Polyunsaturated acids were most affected by chromium, especially those related to chloroplast structures. Ultra-structure studies showed clear thylakoid disorganization in all treated cells. The results indicate that hexavalent chromium affects levels of fatty acids, especially those related to photosynthetic activity.