Paula Mariela González

Exposure to excess dissolved iron in vivo affects oxidative status in the bivalve Mya arenaria

The effect of in vivo Fe exposure on the oxidative metabolism of the bivalve Myaarenaria was studied. Fe was supplemented in natural seawater and resulted in a significant increase in the total Fe content in the bivalve digestive gland (DG) between 9 to 17days of exposure. Mortality of treated animals increased drastically after day 18. Oxidative stress conditions were characterized in DG through assessment of the generation of reactive oxygen species (ROS) and ascorbyl radical (A) content. Both parameters were affected following a biphasic profile showing significant increases by days 2 and 9 of Fe exposure. The content of 2-thiobarbituric acid reactive substances (TBARS) was significantly increased over control values by days 2, 9 and 17 of treatment. The labile Fe pool (LIP) in isolated DG was elevated over control values by day 7, and maintained this increase until day 17 of Fe exposure. The content of NO, assessed by EPR spin trapping, was 60% lower in DG of animals exposed for 2days to Fe than in control values, with no further changes. The biphasic profile of oxidative stress response to Fe exposure in DG suggests that at early stages of Fe supplementation the cellular control mechanisms, such as CAT activity, were operative to limit oxidative damage, but further Fe exposure overwhelmed these abilities. Moreover, the second phase could be understood as the consequence of the exhaustion of cellular protective systems that could also involve NO.

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.

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.

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.

Iron Overload and Lipid Peroxidation in Biological Systems

Fe is an essential element for the growth and well-being of almost all living organisms, except for some strains of lactobacillus, where the role of Fe may be assumed by another metal [1]. It is involved in many biological functions since by varying the ligands to which it is coordinated, Fe has access to a wide range of redox potentials and can participate in many electron transfer reactions, spanning the standard redox potential range. It is also involved in O2 transport, activation, and detoxification, in N2 fixation and in several of the reactions of photosynthesis [2]. However, there are problems in the physiological management of Fe, since in spite of its overall abundance, usable Fe is in short supply because at physiological pH under oxidizing conditions, Fe is extremely insoluble. Anytime Fe exceeds the metabolic needs of the cell it may form a low molecular weight pool, referred to as the labile iron pool (LIP), which catalyzed the conversion of normal by-products of cell respiration, like superoxide anion (O2-) and hydrogen peroxide (H2O2), into highly damaging hydroxyl radical (•OH) through the Fenton reaction (reaction 1) or by the Fe2+ catalyzed Haber-Weiss reaction (reaction 2), or into equally aggressive ferryl ions or oxygen-bridged Fe2+/Fe3+ complexes. Fe3+ can be reduced either by O2(reaction 3) or by ascorbate leading to further radical production.

Cellular Oxidant/Antioxidant Network: Update on the Environmental Effects Over Marine Organisms

Aquatic organisms are exposed and adjust to a wide variety of environmental challenges including natural and anthropogenic factors. Natural sources are understood the effects of temperature, and saline fluctuations, oxygen availability, the relative abundance of chemical elements and pathogenic invasion. On the contrary, anthropogenic factors are considered the availability of heavy metals, the presence of hydrocarbons, industrial and urban wastes, and pesticides. Moreover, these organisms suffer, in order to maintain homeostasis, growth and reproduction, the effect of temporal and spatial variations. All the environmental changes (natural and non-natural) may cause a different degree of stress in aquatic organisms, via induction of disbalance between the generation and elimination of reactive oxygen species and reactive nitrosative species. A brief summary on the actual knowledge on the establishment, by environmental effects, of oxidative/nitrosative stress and the effect on the antioxidant system in marine organisms, is presented in this review to contribute to the deeper understanding of the complexity of the metabolic and physiological changes that aquatic organisms are constantly suffering.

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.

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.

Oxidative status of respiratory tissues of the bivalve Mya arenaria after exposure to excess dissolved iron

The gills and mantle are the first tissues exposed to altered oxidative conditions during dissolved Fe uptake. The aim of this work was to characterize Fe affected oxidative metabolism on the respiratory tissues of Mya arenaria. After 9 days of exposure to elevated Fe levels (500 μM of Fe as Fe-EDTA), a significant increase in the total Fe and labile Fe pool content was found in the gills. Dichlorofluorescein diacetate oxidation rate was higher after 17 days of exposure as compared to controls. Thiobarbituric acid reactive substances content increased 1.9- and 3.7-fold over controls on day 9 and 17, respectively. Both, catalase activity and nitrate and nitrite content decreased significantly on days 9 and 17 compared with controls. Similar effects were observed in mantle, but catalase activity was not affected. The results showed that in respiratory tissues the labile Fe pool is critically controlled to avoid radical-dependent cellular deterioration, but when the endogenous protection mechanisms are overwhelmed, tissue injury was observed.

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.