Fe, oxidative and nitrosative metabolism in the Antarctic limpet Nacella concinna.

The hypothesis of this work was that oxidative and nitrosative metabolism in the digestive gland (DG) of two limpet populations (intertidal and subtidal) of the Antarctic species Nacella concinna show different behavior when they were exposed to either intermittent (intertidal) or constant (subtidal) natural Fe. Total Fe content and labile Fe pool were higher in the DG of the subtidal compared to the intertidal population. However, no significant differences between populations were seen on the Fe atoms content of the isolated ferritin. Ascorbyl radical content was 2.0±0.4 and 6.5±0.8pmol/mg FW in the DG of the intertidal and subtidal animals, respectively. Lipid damage, assessed as content of thiobarbituric reactive substances, was different between the tissues of intertidal and subtidal samples, 491±102 and 1242±367pmol/mg FW, respectively. Catalase and superoxide dismutase activities showed no differences between the limpets. Nitric oxide (NO) content was 25±3 and 22±2pmol/mg FW in DG from intertidal and subtidal animals, respectively. NO synthase-like (NOS-like) activity was evaluated supplementing the samples with the enzyme co-factors, and the inhibitory effect of Nω-nitro-L-arginine methyl ester hydrochloride was tested. NO generation rate was 3.4±0.3 and 4.7±0.6pmol/minmg FW in DG from the intertidal and subtidal population, respectively. These results showed that the oxidative condition of the limpet population constantly covered by the Fe enriched water is more affected than the intertidal population. However, the nitrosative metabolism seems to be independent of the environmental high Fe content since similar NO steady state concentration and NOS-like activity were measured in both populations.

Seasonality and toxins effects on oxidative/nitrosative metabolism in digestive glands of the bivalve Mytilus edulis platensis.

The hypothesis presented here is that oxidative and/or nitrosative metabolism in the bivalve Mytilus edulis platensis is altered by the presence of planktonic toxins. Digestive glands (DG) were isolated from specimens collected in the Argentinean Sea during summer, winter and spring (in the presence of harmful planktonic toxins). The labile iron pool content was not significantly different in DG from animals collected in summer and winter, but was 2.3-fold increased in samples from spring compared to summer collected mollusks. The 2',7' dichlorofluorescein diacetate (DCFH-DA) oxidation, ascorbyl radical/ascorbate and lipid radical/α-tocopherol content ratios showed no significant differences between samples collected in winter and summer. However, spring collected samples showed significantly higher DCFH-DA oxidation rate and oxidative ratios in comparison to DG from mollusks collected in summer. Superoxide dismutase activity decreased by 75% in winter, and 93% in spring, compared to samples collected in summer. Glutathione S-transferase activity decreased by 89% in winter, and 30% in spring, compared to samples collected in summer. Catalase activity in winter animals increased by 3.8-fold in comparison to summer values, with no differences between spring and summer collected mollusks. Nitrite plus nitrate content was not significantly different among samples collected in the three seasons, but nitric oxide content was 8.5- and 2.7-fold higher in samples from winter and spring collected mollusks than values obtained in summer, respectively. These results showed the lack of effects of climatic changes on the integrative oxidative indexes; however, under exposure to toxins, both oxidative and nitrosative metabolisms were affected.

Electronic paramagnetic resonance (EPR) for the study of ascorbyl radical and lipid radicals in marine organisms.

Electron paramagnetic resonance (EPR) spectroscopy detects the presence of radicals of biological interest, such as ascorbyl radical (A(•)) and lipid radicals. A(•) is easily detectable by EPR even in aqueous solution at room-temperature. Under oxidative conditions leading to changes in total ascorbate (AH(-)) content, the A(•)/AH(-) ratio could be used to estimate early oxidative stress in the hydrophilic milieu. This methodology was applied to a wide range of aquatic systems including algae, sea urchin, limpets, bivalves and fish, under physiological and oxidative stress conditions as well. The A(•)/AH(-) ratio reflected the state of one part of the oxidative defense system and provided an early and simple diagnosis of environmental stressing conditions. Oxidative damage to lipids was assessed by the EPR-sensitive adduct formation that correlates well with cell membrane damage with no interference from other biological compounds. Probe instability, tissue metabolism, and lack of spin specificity are drawback factors for employing EPR for in vivo determination of free radicals. However, the dependability of this technique, mostly by combining it with other biochemical strategies, enhances the value of these procedures as contributors to the knowledge of oxidative condition in aquatic organisms.

Domoic Acid Oxidative Effects on the Microalgae Phaeodactylum tricornutum.

Domoic acid (DA) is a natural occurring marine biotoxin. Oxidative stress generation due to DA exposure was reported in animals, but little is known on the phytoplankton community. The aim of this work was to verify whether exposure to DA in the marine diatom Phaeodactylum tricornutum favors reactive oxygen species (ROS) generation in the intracellular environment modifying its antioxidant capacity. Active species production, non-enzymatic antioxidant content, and antioxidant enzyme activities over the three growth phases of P. tricornutum exposed to 64 µM DA were evaluated. Results obtained in exponential growing cells showed a time-depending seven-fold increase in the 2',7' dichlorofluorescein diacetate dye oxidation rate. Superoxide dismutase and catalase activities showed a two-fold increase, and glutathione related enzymes activities were also significantly increased in treated diatoms as compared to controls. However, glutathione and ascorbate contents significantly decreased after incubation of the cells with DA. Similar effects were observed in latent and stationary phases of cell development. These results showed that DA could cause a severe oxidant-dependent impact on a non-toxic algae.

Possible role of seasonality and harmful algal blooms (HAB) on the oxidative and nitrosative metabolisms in hemocytes.

Bivalves survive to biotoxin consumption but their metabolism could be affected. The objective of this work was to study the oxidative and nitrosative changes in the hemocytes of the mussel Mytilus edulis platensis in different seasons, including spring, characterized by the appearance of harmful algal blooms (HAB). Reactive species generation rate (measured as the 2',7'dichlorofluorescein diacetate oxidation rate) was increased by 2.5- and 8.3-fold in hemocytes from spring and summer, respectively, as compared to winter samples. Neither total Fe nor labile Fe pool content was changed in the three seasons. Superoxide anion generation rate was 3-fold higher in spring as compared to winter and summer samples. Catalase content in spring cells were significantly higher as compared to winter (60%) and summer (3-fold increase) but glutathione-S-transferase activity only increased compared to summer season (125% increase). Lipid radical content in spring samples was 140 and 50% higher as compared to cells from winter and summer, respectively. Nitric oxide and nitro-tyrosine content were significantly higher in samples from spring as compared to values obtained either in winter or summer cells. Considering the aspects that influence metabolism, changes in temperature seem to mainly affect the oxidative over the nitrosative condition of the hemocytes. Nevertheless, HAB biotoxins seem as a contributing factor to affect not only reactive oxygen species generation, antioxidant activity and protein/lipid damage, but also the nitrosative metabolism. In this regard, the changes in the nitric oxide content are new and critical evidence that HAB-related toxins could affect reactive nitrogen species metabolism.

Iron and radical content in Mya arenaria. Possible sources of NO generation.

The objective of this work was to analyze oxidative metabolism in Mya arenaria. Total Fe content in M. arenaria collected in the German Wadden Sea was 1.9+/-0.7, 0.7+/-0.1 and 0.17+/-0.01 nmol/mg fresh weight (FW), in digestive glands (DG), mantle and gills, respectively. Labile Fe pool, assessed by electronic paramagnetic resonance (EPR), was 146+/-10 pmol/mg FW, and by the fluorescence method employing calcein it was 118+/-9 pmol/mg FW. The lipid radical content in the DG, assessed by EPR, was 27+/-7 pmol/mg FW, and the thiobarbituric reactive substances content amounted to 57+/-8 pmol/mg FW. Ascorbyl radical (A) content, assessed by quantification of EPR signals, was 0.04+/-0.01 pmol/mg FW, and the ascorbate content (AH(-)) was 478+/-12 pmol/mg FW. The ratio A/AH(-) was (8+/-1)x10(-5)AU, suggesting a minimum oxidative stress even under physiological conditions, presumably depending on basal metabolic functions. The content of nitric oxide (NO), assessed by EPR, was 99+/-3 pmol/mg FW. The generation rate of NO by nitric oxide synthase-like activity (NOS-like) was assayed as NO production detected by EPR in the presence of l-arginine and NADPH, and was 3.16+/-0.06 pmol/(mg FW min). The data presented here document the detectable presence of highly reactive species in M. arenaria.

Detection of Nitric Oxide via Electronic Paramagnetic Resonance in Mollusks.

Electronic paramagnetic resonance (EPR) is an appropriate tool to identify free radicals formed in tissues under normal as well as stressful conditions. Since nitric oxide (NO) as a free radical has paramagnetic properties it can be detected by EPR. The use of spin traps highly improves the sensitivity allowing NO identification, detection and quantification at room temperature in vitro and in vivo conditions. NO production in animals is almost exclusively associated to an enzyme family known as Nitric Oxide Synthases (NOSs). The digestive glands of mollusks are a major target for oxidative disruption related to environmental stress. A simple EPR-methodology to asses both, the presence of NO and its rate of generation in tissues from different mollusk species, is reported here.

Hypoxically Induced Nitric Oxide: Potential Role as a Vasodilator in Mytilus edulis Gills.

Intertidal Mytilus edulis experience rapid transgression to hypoxia when they close their valves during low tide. This induces a physiological stress response aiming to stabilize tissue perfusion against declining oxygen partial pressure in shell water. We hypothesized that nitric oxide (NO) accumulation supports blood vessel opening in hypoxia and used live imaging techniques to measure NO and superoxide anion ( O 2 ∙ - ) formation in hypoxia-exposed gill filaments. Thirty minutes of moderate (7 kPa pO2) and severe hypoxia (1 kPa pO2) caused 1.6- and 2.4-fold increase, respectively, of NO accumulation in the endothelial muscle cells of the hemolymphatic vessels of the gill filaments. This led to a dilatation of blood vessel diameter by 43% (7 kPa) and 56% (1 kPa), which facilitates blood flow. Experiments in which we applied the chemical NO-donor Spermine NONOate (concentrations ranging from 1 to 6 mM) under normoxic conditions corroborate the dilatational effect of NO on the blood vessel. The formation of O 2 ∙ - within the filament epithelial cells increased 1.5 (7 kPa) and 2-fold (1 kPa) upon treatment. Biochemical analysis of mitochondrial electron transport complexes in hypoxia-exposed gill tissue indicates decreased activity of complexes I and III in both hypoxic conditions; whereas complex IV (cytochrome-c oxidase) activity increased at 7 kPa and decreased at 1 kPa compared to normoxic exposure conditions. This corresponds to the pattern of pO2-dependent gill respiration rates recorded in ex-vivo experiments. Severe hypoxia (1 kPa) appears to have a stabilizing effect on NO accumulation in gill cells, since less O2 is available for NO oxidation to nitrite/nitrate. Hypoxia thus supports the NO dependent inhibition of complex IV activity, a mechanism that could fine tune mitochondrial respiration to the local O2 availability in a tissue. Our study highlights a basal function of NO in improving perfusion of hypoxic invertebrate tissues, which could be a key mechanism of tolerance toward environmental O2 variations.

A kinetic approach to assess oxidative metabolism related features in the bivalve Mya arenaria.

Electron paramagnetic resonance uses the resonant microwave radiation absorption of paramagnetic substances to detect highly reactive and, therefore, short-lived oxygen and nitrogen centered radicals. Previously, steady state concentrations of nitric oxide, ascorbyl radical (A·) and the labile iron pool (LIP) were determined in digestive gland of freshly collected animals from the North Sea bivalve Mya arenaria. The application of a simple kinetic analysis of these data based on elemental reactions allowed us to estimate the steady state concentrations of superoxide anion, the rate of A· disappearance and the content of unsaturated lipids. This analysis applied to a marine invertebrate opens the possibility of a mechanistic understanding of the complexity of free radical and LIP interactions in a metabolically slow, cold water organism under unstressed conditions. This data can be further used as a basis to assess the cellular response to stress in a simple system as the bivalve M. arenaria that can then be compared to cells of higher organisms.

Iron and nitrosative metabolism in the Antarctic mollusc Laternula elliptica.

The objective of this work was to study Fe distribution, and oxidative and nitrosative metabolism in Laternula elliptica for physiological analysis and interspecific comparisons. Lipid peroxidation, superoxide dismutase and catalase activity and total Fe content were estimated in the digestive glands (DG) of L. elliptica. The labile Fe pool (LIP) represents the amount of cellular Fe responsible for catalyzing radical-dependent reactions. LIP assessed by the calcein assay, represents 3.5% of the total Fe in L. elliptica. Experimental isolation of ferritin (Ft) was performed. Subunit analyses of the protein by SDS-polyacrilamide gel electrophoresis indicated that the protein was composed of 20.6kDa protein subunits, consistent with the horse spleen Ft and the molecular weight markers, however, a higher molecular mass subunit could appear. The identity of the protein was confirmed by Western blot analysis. The nitrate+nitrite content was 73±7pmol/mg fresh mass (FW). The nitric oxide (NO) content in DG homogenates, assessed by electronic paramagnetic resonance (EPR) spin trapping measurements using the NO trap sodium-N-methyl-D-glucamine dithiocarbamate-Fe at room temperature, was 30±2pmol/mg FW. Nitric oxide synthase-like activity (1.50±0.09pmol/mg FW min) was assessed by measuring NO production by EPR in the presence of L-arginine (L-A) and NADPH. This activity was significantly inhibited by L-A analogs such as Nω-nitro-L-arginine methyl ester hydrochloride (-77%) and Nω-nitro-L-arginine (-62%), or by the lack of added L-A (-55%). The data presented here documented the physiological presence of labile Fe, Ft and highly reactive nitrogen species, and are the first evidence that support the hypothesis that NO being generated in L. elliptica might contribute to restrict oxidative damage by a close link with Fe metabolism.