Gabriela Malanga

Iron-dependent oxidative stress in Chlorella vulgaris

Abstract The role of iron as catalyst for oxidative stress was studied during the development of Chlorella vulgaris cells. Increases on iron availability beyond 200 μM led to a decrease in the growth rate of the cultures of C. vulgaris. Quantification of the EPR signals of POBN/lipid radicals adducts indicated that iron addition increased lipid radical content in the membranes (more than three-fold at 500 μM Fe). The ratio of ascorbyl radical content/ascorbate content was increased in cells exposed to 500 μM iron as compared to standard conditions (90 μM Fe). β-Carotene content was not affected by iron addition, however, α-tocopherol, ascorbate and total thiol content in C. vulgaris cells was increased when iron supply was higher than 90 μM. The morphology of the cells developed under 500 μM iron showed a decreased number of ribosomes and alterations in the cellular starch deposits as compared to the cells grown at 90 μM iron. Exposure to UV-B increased the content of lipid radicals and affected cellular internal structure as compared to non-irradiated cells developed at 500 μM iron. Even though results from model systems cannot be directly used to predict ecological effects, these data suggest that oxidative stress triggered by an excess of iron could affect cellular growth and have a negative impact to phytoplankton.

N-acetylcysteine-dependent protection against UV-B damage in two photosynthetic organisms

Cellular thiols, specially glutathione, appear to play a key role in protection against oxidative damage arising from a number of stress conditions. Thus, the role of N-acetylcysteine (NAC) as a protector against oxidative damage associated with ultraviolet B (UV-B) in two photosynthetic organisms was evaluated. Algal cultures (Chlorella6ulgaris) and soybean leaves (Glycine max var. Hood) were supplemented with 1 mM NAC before irradiation. The content of total thiols was significantly increased after treatment. After exposure to UV-B, the ascorbyl radical-dependent electron paramagnetic resonance (EPR) signal was enhanced by 76 and 46% and lipid radical-dependent EPR signal increased by 83 and 188% in algal cultures and soybean leaves, respectively. Treatment with 1 mM NAC kept ascorbyl and lipid radical content in algae and soybean leaves exposed to UV-B at the basal level. Supplementation with 1 mM NAC did not affect the content of lipid-soluble antioxidants (a-tocopherol, b-carotene) in C.6ulgaris or in soybean leaves. UV-B irradiation increased catalase activity in the algae by 145% and in soybean leaves by 34%, while total superoxide dismutase (SOD) activity was not affected. In both photosynthetic organisms the increase in catalase activity after UV-B exposure did not occur in the presence of 1 mM NAC. The results suggest that thiols play an important role in triggering cellular control against UV-B-related damage, as NAC pretreatment significantly decreased UV-B-dependent radical generation after irradiation.

UV-B effects on Antarctic Chlorella sp cells

Growth of Antarctic Chlorella sp cells was measured in cultures irradiated with 30 kJ m(-2) UV-B (280-320 nm). The specific growth rate immediately after the lag phase was 0.36+/-0.06 and 0.26+/-0.03 day(-1) for unirradiated cultures and cultures irradiated with UV-B, respectively, UV-B irradiation significantly decreased ascorbate content by 54.5%, and increased the ascorbyl radical content/ascorbate content ratio by 2.25-fold in algae cultures in log phase. UV-B exposure significantly decreased by 95, 62 and 71% the content of alpha-tocopherol, beta-carotene and total thiols, respectively, in cells in log phase of development. The cellular content of alpha-tocopherol, beta-carotene and total thiols was reduced by 78, 43 and 44%, respectively in stationary phase, as compared to the antioxidant content in the cells during log phase of development. UV-B exposure reduced the content of alpha-tocopherol and total thiols in stationary phase of development by 64 and 91%, respectively, as compared to unirradiated cells. The content of beta-carotene in stationary phase was not affected by UV-B exposure. The results presented here suggest that increased UV-B radiation was responsible for the development of oxidative stress conditions, assessed as the ascorbyl radical content/ascorbate content ratio, in Antarctic Chlorella sp cells. Moreover, a significant decrease in the content of both lipid and water soluble antioxidants might contribute to establish oxidative stress in the cells.

Seasonal and pollution-induced variations in biomarkers of transplanted mussels within the Beagle Channel.

The occurrence of biomarker variations linked to environmental factors makes it difficult to distinguish the effect of pollution. In an attempt to evaluate spatial and seasonal effects of environmental parameters on biomarker responses, mussels Mytilus edulis chilensis coming from an aquaculture farm were transplanted to several points within Ushuaia Bay (Beagle Channel) for 6 weeks in summer and winter. Activities of superoxide dismutase, catalase, glutathione-S-transferase and levels of lipid peroxidation were measured in gills and digestive gland. Cu, Zn, Fe, Cd and Pb concentrations were also assessed. Results indicated a significant effect of seasons on biological responses as well as in metal bioaccumulation showing the influence of natural factors such as dissolved oxygen, temperature and food availability. The interdependence of those environmental factors is important for the homeostasis of thermoconformers, especially regarding their oxidative metabolism and should also be taken into consideration to distinguish natural from pollution-induced variations.

Biomarkers of environmental stress in gills of ribbed mussel Aulacomya atra atra (Nuevo Gulf, Northern Patagonia)

In this study, we assessed in gills of native ribbed mussels Aulacomya atra atra from three sites within Nuevo Gulf (Northern Patagonia) several biomarkers such as reactive oxygen species (ROS), lipid radicals (LR), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and metallothionein (MT). Furthermore, concentrations of main trace metals (Fe, Al, Zn, Cu, Cd and Pb) were quantified in mussel tissue. Results showed significant induction of SOD, GST, MT and MDA, as well as, higher concentration of Fe, Al and Cd in winter than in summer. The high MDA content measured in mussels from Folías Wreck seemed to be caused by the very high levels of Fe that would come from the corrosion of the vessel. Mussels from the control site Punta Cuevas presented the lowest levels of Cd and the highest of Al in winter. Despite positive correlations were found between Al and GST and MT, no spatial differentiation was detected in those biomarkers. On the other hand, MT was only related to Al been most likely influenced by environmental variables than by the trace metals. It has to be highlighted that the relationship detected among water temperature, nutrients and antioxidant responses in gills is probably related to the fact that this tissue is in direct contact with water and it is sensitive to its fluctuations. Taking into account that mussel gill is a tissue actively proliferating and the first target of contaminants present in water, so that changes in its antioxidant system can provide an earlier warning signal than in other tissues.

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.

Assessment of antioxidant responses and trace metal accumulation by digestive gland of ribbed mussel Aulacomya atra atra from Northern Patagonia

Seasonal and spatial variability of trace metal concentrations and of a battery of antioxidant parameters were evaluated in digestive gland of the ribbed mussel Aulacomya atra atra. Fe, Al and Cu accumulated in tissue exhibited maximum values in winter, coinciding partially with the highest labile concentrations of Fe and Cu in sediment. Metals, as other pollutants, are known to influence the oxidative status of organisms and antioxidant enzymes have been often proposed as biomarkers of contaminant effects. Seasonal variations of trace metals did not appear to influence those of biochemical parameters, which generally showed an opposite trend with higher enzymatic activities in summer when trace metal concentrations were lower. Organisms from Punta Cuevas (control site) showed higher induction of reactive oxygen species production than those from both considered impacted sites, suggesting the possibility of some biochemical adaptation in organisms or a higher modulation of environmental and physiological factors on antioxidant responses than levels of trace metals. This study, which is the first in the area in this matter, showed that seasonal variations of potential biomarkers should be incorporated into interpretation of long-term biomonitoring studies in this marine coastal ecosystem.

Antioxidant Defenses and Trace Metal Bioaccumulation Capacity of Cymbula nigra (Gastropoda: Patellidae)

The present study deals with the effect of trace metals on the endangered limpet Cymbula nigra. The Bay of Algeciras (Strait of Gibraltar) was used as the study site. Important industrial activity takes place in the area, including frequent oil spills. However, it is home to important populations of C. nigra. The objective of this work was to determine if these animals were being affected at a subcellular level by the pollutants present in their environment and to analyze the trace metal concentrations in the animal’s soft tissues. To determine the effects of water quality on the antioxidant activity and concentrations through field experimentation, a total of six sites were selected in Algeciras Bay, three located in the inner areas (environmentally degraded sites with higher levels of pollutants) and three in the outermost areas of the Bay. Stress associated to reactive oxygen species formation was assessed on digestive glands and gills as the enzymatic antioxidant activity of catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST) and as the concentrations of lipid-soluble (α-tocopherol and β-carotene) and the water-soluble antioxidants (reduced and oxidized glutathione (GSH and GSSG)). Gills and digestive glands of those animals located in the inner areas of Algeciras Bay showed higher CAT activity values than those located in the outer areas. As a general pattern, we observed higher antioxidant activities and concentrations in digestive glands that in gills, suggesting the possibility that pollutants are mainly being incorporated by limpets through the food. As a general rule, larger animals showed greater concentrations of these compounds. Iron, zinc, and manganese, in this order, were present in the tissues at the highest concentrations. Chromium and manganese were found in significantly higher concentrations in those animals collected from the inner areas of the Bay. Through the present study, we provide the first data regarding the antioxidant defense levels and metal accumulation capacity of this species, and we reinforce the idea that this endangered species may be, in fact, relatively tolerant to degraded environments.

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.

Metals from mine waste as potential cause of oxidative stress in burrowing crab Neohelice granulata from San Antonio bay.

The Natural Protected Area San Antonio bay is of particular importance for its congregation of migratory shorebirds and it has been declared one of the Western Hemisphere Shorebird Reserve Network International site (WHSRN). Present study represents the first assessment of variation on oxidative stress biomarkers in male crab Neohelice granulata from San Antonio bay (Río Negro, Argentina) under field conditions, associated mainly to metal contamination coming from passive mining wastes. Three sites were sampled once every three months from November 2012 to August 2013 within this sea inlet (Pile, Fishery and Port) and a control site at the southeast of the bay (Punta Perdices). Accumulation of Ni, Zn, Cr and Al varied only with seasons although without a constant trend, meanwhile Cd, Cu and Pb also varied among sites being highest in Pile and Port. Biochemical results indicated that variations in catalase activity was only site specific being maximum in Pile; meanwhile lipid radical, α-tocopherol and metallothioneins were only seasonal specific being higher in autumn and winter. Seasonal variation was also found for total thioles, being the content higher in summer and autumn than in winter. Correlation analysis revealed that malondialdehyde and α-tocopherol have a positive association with Al and negative with Ni, meanwhile GST has a positive association with Fe. Crabs from the closest area to the waste pile did not exhibit a differentiated oxidative pressure despite the higher accumulation of metals. It is possible that crabs from contaminated areas have developed a tolerance to metals, indicating a strong ecotoxicological selective pressure. More studies are needed to assess whether there is a transfer of metals through the food chain.