How water acidification influences the organism antioxidant capacity and gill structure of Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) at normoxia and hypoxia.

The effect of water acidification in combination with normoxia or hypoxia on the antioxidant capacity and oxidative stress markers in gills and hemolymph of the Mediterranean mussel (Mytilus galloprovincialis), as well as on gill microstructure, has been evaluated through an in vivo experiment. Mussels were exposed to a low pH (7.3) under normal dissolved oxygen (DO) conditions (8 mg/L), and hypoxia (2 mg/L) for 8 days, and samples were collected on days 1, 3, 6, and 8 to evaluate dynamic changes of physiological responses. Cytoplasmic concentrations of reactive oxygen species (ROS) and levels of DNA damage were measured in hemocytes, while the activity of catalase (CAT) and superoxide dismutase (SOD) and histopathological changes were assessed in gills. The results revealed that while water acidification did not significantly affect the activity of SOD and CAT in gills under normoxic and hypoxic conditions, there was a trend towards suppression of CAT activity at the end of the experimental period (day 8). Similarly, we did not observe increased formation of ROS in hemocytes or changes in the levels of DNA damage during the experimental period. These results strongly suggest that the oxidative stress response system in mussels is relatively stable to experimental conditions of acidification and hypoxia. Experimental acidification under normoxia and hypoxia caused changes to the structure of the gills, leading to various histopathological alterations, including dilation, hemocyte infiltration into the hemal sinuses, intercellular edema, vacuolization of epithelial cells in gill filaments, lipofuscin accumulation, changes in the shape and adjacent gill filaments, hyperplasia, exfoliation of the epithelial layer, necrosis, swelling, and destruction of chitinous layers (chitinous rods). Most of these alterations were reversible, non-specific changes that represent a general inflammatory response and changes in the morphology of the gill filaments. The dynamics of histopathological alterations suggests an active adaptive response of gills to environmental stresses. Taken together, our data indicate that Mediterranean mussels have a relative tolerance to water acidification and hypoxia at tissue and cellular levels.

Accumulation, functional and antioxidant responses to acute exposure to Di(2-ethylhexyl)phthalate (DEHP) in Mytilus galloprovincialis.

Mussels were exposed to di-(2-ethylhexyl) phthalate (DEHP) (0.4 mg L-1 and 4.0 mg L-1) for 24 h and 48 h and its effect on hemocyte cellular composition and spontaneous reactive oxygen production (ROS) levels in hemocytes were evaluated. Exposure to DEHP induced a loss in spontaneous ROS production levels in hemocytes and decreased agranulocyte number in hemolymph. DEHP was found to accumulate in hepatopancreas of mussels and this process was associated with an increase of catalase (CAT) activity after 24 h incubation. At the end of the experimental period (48 h) CAT activity recovered up to control levels. Superoxide dismutase (SOD) activity in hepatopancreas increased following 48 h exposure to DEHP. The results indicated that DEHP could affect hemocyte immune properties, and also cause non-specific general stress-response of the antioxidant complex, which, in turn, was not associated with pronounced oxidative stress.

Functional changes in hemocytes and antioxidant activity in gills of the ark clam Anadara kagoshimensis (Bivalvia: Arcidae) induced by salinity fluctuations.

This study describes the analysis of antioxidant enzymatic activities (catalase and superoxide dismutase) in gills and functional state of hemocytes (osmotic stability, mitochondrial membrane potential) of ark clams (Anadara kagoshimensis) from the Black Sea basin exposed to salinity stress. For this, the effects of 48 h periods of exposure to low (8 ‰, 14 ‰) and high (35 ‰, 45 ‰) salinity were assessed. Our results showed that ark clams, A. kagoshimensis, possessed pronounced tolerance to hypersalinity stress and are sensitive to a short-time hyposalinity treatment. Salinity 35 ‰ inhibited production of reactive oxygen species (ROS) by hemocytes and did not affect their levels of mitochondrial membrane potential. Acclimation to 45 ‰ salinity caused significant increase in mitochondrial membrane potential accompanied with recovery of intracellular ROS levels up to controls levels. Acclimation to low salinity (8 ‰) induced an increase in both ROS and mitochondrial membrane potential levels in hemocytes. Catalase and superoxide dismutase activity in gills decreased following acclimation to low (8 ‰) and high (35 ‰) salinity. Exposure to the highest salinity levels (45 ‰) led to a decrease of superoxide dismutase activity levels, but did not influence the levels of catalase activity. Acclimation to low and high salinity was not accompanied with changes in osmotic fragility of hemocytes despite osmotic fragility curve according to changes in hemolymph osmolarity. Based upon these results, we postulate the involvement of cellular osmoregulatory mechanisms in the adaptation of the ark clam to short-term fluctuations of environmental salinity.

Adaptive potential of the Mediterranean mussel Mytilus galloprovincialis to short-term environmental hypoxia.

Environmental hypoxia naturally occurs in coastal ecosystems and bivalve mollusks have to frequently face fluctuations of dissolved oxygen concentrations. Exposure to hypoxia is often associated with the change of the antioxidant and functional status in bivalves, and restoration of the normal oxygen supply is considered to induce oxidative stress in tissues of mollusks. The study investigates changes in the activity of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), as well as the expression level of SOD and CAT genes in gills of the Mediterranean mussel, Mytilus galloprovincialis, under exposure to low dissolved oxygen concentration (2.2 mg L-1) for 24 h and 72 h, and 24 h reoxygenation period. We also evaluated the intracellular level of reactive oxygen species (ROS), mortality and changes in mitochondrial membrane potential in hemocytes following hypoxia-reoxygenation cycle. 24 h exposure to hypoxia significantly decreased activity of both enzymes, which then recovered up to control levels at the end of 72 h experimental period for SOD and after reoxygenation for CAT. Expression of antioxidant enzyme genes was up-regulated following the 72 h hypoxic exposure period and returned to the basal normoxic level after 24 h reoxygenation. Hypoxia demonstrated a time-dependent effect on the functional state of hemocytes. The 24 h exposure period did not influence aerobic respiration of hemocytes, but prolonged hypoxia (72 h) was associated with a substantial decrease in mitochondrial membrane potential of hemocytes. The intracellular ROS level and mortality of hemocytes did not change under hypoxia. Reoxygenation period was accompanied with a significant decrease of intracellular ROS level. This study indicated that hypoxia did not induce the pronounced oxidative stress in gills and the changes in the antioxidant status were reversible within 24 h of reoxygenation. Hemolymph demonstrated a stable functional state indicating the tolerance of mussels to short-time hypoxia.

Acute hypoxic exposure: Effect on hemocyte functional parameters and antioxidant potential in gills of the pacific oyster, Crassostrea gigas.

Bivalve mollusks are frequently subjected to fluctuations of dissolved oxygen concentration in the environment which can represent a significant threat to bivalve antioxidant status. In this work the effects of hypoxia on hemocyte reactive oxygen species (ROS) production and level of mitochondrial potential as well as the activity and expression level of catalase (CAT) and superoxide dismutase (SOD) in gills of Crassostrea gigas were investigated after 24 h and 72 h exposure. 24 h hypoxia promoted an increase of mitochondrial membrane potential in agranulocytes and induced ROS accumulation in granulocytes. 72 h exposure substantially decreased hemocyte mitochondrial potential and intracellular ROS level in all hemocyte types. No significant changes in the activity of CAT in gills were observed following both 24 h and 72 h exposure periods compared to control. SOD activity in gills decreased after 72 h exposure to hypoxia but did not change under 24 h hypoxia. Significant up-regulation of SOD gene and no changes in expression level of CAT were observed in all experimental groups. The results indicate an overall shift in antioxidant status in gills and hemocytes of the Pacific oyster that may act as compensatory mechanisms to maintain redox homeostasis after a short-term (24 h) exposure and represent the occurrence of oxidative stress conditions at the end of 72 h hypoxia.

[ELEMENTS OF THE LOW MOLECULAR WEIGHT CHAIN OF ANTIOXIDANT DEFENSE IN TISSUES OF THE BLACK SEA MOLLUSC ANADARA KAGOSHIMENSIS BRUGÙIERE].

The content of reduced glutathione (GSH) and the activity of the coupled with it antioxidant enzymes - glutathione peroxidase and glutathione reductase as well the level of glucose, carbamide and amino acids were investigated in the hepatopancreas, gills and foot of the Black, Sea mollusk Anadara kagoshimensis. The highest content of GSH and the highest activity of glutathione peroxidase were found in mollusk foot, evidencing the active antioxidant role of glutathione played both within composition of this enzyme and independently. The maximal content of glucose, amino acids and carbamide was in the hepatopancreas and gills and the minimal - in the anadara's foot. The possible involvement and role of these low molecular weight antioxidants in the defense of mollusk tissues against action of free radical oxidation and in providing adaptation reactions of anadara in hypoxic habitats are considered. Key words: antioxidant complex, glutathione, glucose, carbamide, amino acids, anadara Anadara kagoshimensis, Black Sea.

[Functional states of the antioxidant enzymatic complex of tissues of Mytilus galloprovincialis Lam. under conditions of oxidative stress].

There are generalized materials on reaction of enzymatic antioxidant (AO) complex of tissues of molluscs of the genus Mytilus to oxidative loading of different intensity. It is shown that with increase of level of oxidative stress, a rise of activity of glutathione peroxidase (GP) and glutathione reductase (GR) is observed, as well as the rate of glutathione (GSH) in tissues also increases. Then the toxic loading is compensated by a rise of activities of enzymes of low affinity to hydroperoxides, specifically of catalase. In the glutathione system, it is possible to identify several relatively steady states: of low, moderate, and highly intensive processes. In several tissues, functioning of the AO system seems to be directed not to rendering harmless, but to generation of reactive oxygen species (O2-, superoxide dismutase), which is likely to be determined by specificity of their running processes (destruction of damaged byssus theads). The molluscan AO complex is characterized by high lability and sensitivity both to physiological states (spawning) and to action of factors of anthropogenic nature (cationic detergents). Reactions of AO complex of the mussel to the natural states and to toxic action are comparable between each other, which is necessary to be taken into account at diagnostics of the aquatic medium.