Oxidative stress in the bivalve Diplodon chilensis under direct and dietary glyphosate-based formulation exposure.

The aim of this study was to evaluate and compare the effects on biochemical parameters and organosomatic indices in the freshwater bivalve Diplodon chilensis exposed to a glyphosate-based formulation under direct and dietary exposures (4 mg a.p./L). After 1, 7, and 14 days of exposure, reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) levels and the activities of glutathione-S- transferase (GST), superoxide dismutase (SOD), and catalase (CAT) were evaluated in the gills and digestive gland. The hepatosomatic (HSI) and branchiosomatic (BSI) indices were also analyzed. Direct and dietary glyphosate-based formulation exposure altered the redox homeostasis in the gills and digestive gland throughout the experimental time, inducing the detoxification response (GST), the antioxidant defenses (SOD, CAT, GSH), and causing lipid peroxidation. After 14 days of exposure, the HSI and BSI increased significantly (43% and 157%, respectively) only in the bivalves under direct exposure. Greater changes in the biochemical parameters were induced by the dietary exposure than by the direct exposure. Furthermore, the gills presented an earlier response compared to the digestive gland. These results suggested that direct and dietary exposure to a glyphosate-based formulation induced oxidative stress in the gills and digestive glands of D. chilensis. Thus, the presence of glyphosate-based formulations in aquatic ecosystems could represent a risk for filter-feeding organisms like bivalves.

Corrigendum: Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves.

[This corrects the article on p. 100 in vol. 9, PMID: 29527172.].

Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves.

Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (<0.2 mg O2/L), hypoxia (2 mg O2/L), and normoxia (9 mg O2/L). Specifically, we investigated the expression of an alternative oxidase (AOX) pathway assumed to shortcut the regular mitochondrial electron transport system (ETS) during metabolic rate depression (MRD) in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP) and succinate dehydrogenase (SDH) in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH) activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx) expression] and apoptotic intensity (caspase 3/7 activity) decreased, whereas an unfolded protein response (HSP90) was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks.

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.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques.

BACKGROUND: Reactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues. Most analytical work uses tissue homogenates, and lacks spatial information on the tissue specific sites of actual ROS formation. Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes at cellular level. RESULTS: Together with oxidative stress measurements, here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of ROS and RNS formation in the living tissue. Our results indicate that a) mitochondria located in the basal parts of the epithelial cells close to the blood vessels are hyperpolarized with high Δψm, whereas b) the peripheral mitochondria close to the cilia have low (depolarized) Δψm. These mitochondria are densely packed (mitotracker Deep Red 633 staining), have acidic pH (Ageladine-A) and collocate with high formation of nitric oxide (DAF-2DA staining). NO formation is also observed in the endothelial cells surrounding the filament blood sinus. ROS (namely H2O2, HOO(•) and ONOO(-) radicals, assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. On the ventral bend of the gills, subepithelial mucus glands contain large mucous vacuoles showing higher fluorescence intensities for O2 (•-) than the rest of the tissue. Whether this O2 (•-) production is instrumental to mucus formation or serves antimicrobial protection of the gill surface is unknown. Cells of the ventral bends contain the superoxide forming mucocytes and show significantly higher protein carbonyl formation than the rest of the gill tissue. CONCLUSIONS: In summary, ROS and RNS formation is highly compartmentalized in bivalve gills under unstressed conditions. The main mechanisms are the differentiation of mitochondria membrane potential and basal ROS formation in inner and outer filament layers, as well as potentially antimicrobial ROS formation in the central blood vessel. Our results provide new insight into this subject and highlight the fact that studying ROS formation in tissue homogenates may not be adequate to understand the underlying mechanism in complex tissues.

Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D. chilensis, continuous and long term feeding with E. gracilis cells favors immune response and reduces the damage caused by sewage pollution exposure on hemocytes. Nevertheless, diet and transplantation procedures may produce negative effects on the oxidative balance of gills and digestive gland and should be taken into account for bioremediation strategies.

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Biomarker responses to sewage pollution in freshwater mussels (Diplodon chilensis) transplanted to a Patagonian river.

Field and laboratory experiments were combined to evaluate biomarker responses of Diplodon chilensis to sewage pollution. Mussels from an unpolluted area in Lacar lake (S0) were caged at a reference site (S1) and at two sites with increasing sewage pollution (S2, S3) in Pocahullo river (all in Argentina). After 1 month, gill (g) glutathione S-transferase (GST) and catalase (CAT) activities, and lipid peroxidation (TBARS) were found to be significantly elevated in S3, gGST being positively correlated with fecal bacteria (FC) concentration. Digestive gland (dg) enzyme activities were depressed and dgTBARS were increased in all transplanted mussels. After 3 mo, most variables returned to control levels in S1 mussels except for dgCAT and dgTBARS. After seven months, GST and CAT activities of S0 and S3 mussels were evaluated in the laboratory, before and after acute exposure (8 h) to high fecal bacteria concentration ([FC] in S3x 2). gGST increased in both groups, while dgGST responded only in S3 mussels. gCAT and dgCAT activities were similarly increased by acute exposure in both groups. Our results suggest that gGST and gCAT are suitable biomarkers for high FC pollution regardless of previous exposure history. In addition, we show that dgCAT is sensitive to the acute increase in FC load, both in naive and long-term exposed individuals, while dgGST becomes responsive after long-term acclimatization.

Health status and bioremediation capacity of wild freshwater mussels (Diplodon chilensis) exposed to sewage water pollution in a glacial Patagonian lake.

Deleterious effects on health and fitness are expected in mussels chronically exposed to sewage water pollution. Diplodon chilensis inhabiting SMA, an area affected by untreated and treated sewage water, shows increased hemocyte number and phagocytic activity, while bacteriolytic and phenoloxidase activities in plasma and reactive oxygen species production in hemocytes are lower compared to mussels from an unpolluted area (Yuco). There are not differences in cell viability, lysosomal membrane stability, lipid peroxidation and total oxygen scavenging capacity between SMA and Yuco mussels' hemocytes. Energetic reserves and digestive gland mass do not show differences between groups; although the condition factor is higher in SMA than in Yuco mussels. Gills of SMA mussels show an increase in mass and micronuclei frequency compared to those of Yuco. Mussels from both sites reduce bacterial loads in polluted water and sediments, improving their quality with similar feeding performance. These findings suggest that mussels exposed to sewage pollution modulate physiological responses by long-term exposure; although, gills are sensitive to these conditions and suffer chronic damage. Bioremediation potential found in D. chilensis widens the field of work for remediation of sewage bacterial pollution in water and sediments by filtering bivalves.

Effects of sewage discharges on lipid and fatty acid composition of the Patagonian bivalve Diplodon chilensis.

Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clams (Dipolodon chilensis), from a sewage-polluted (SMA) and a clean site, were compared. Trophic markers FA were analyzed in clams and sediment. Saturated FA (SAFA), and bacteria and sewage markers were abundant in SMA sediments, while diatom markers were 50% lower. Proportions of SAFA, branched FA, 20:5n-3 (EPA) and 22:6n-3 (DHA) were higher in SMA clams. Chronic exposure of D. chilensis to increasing eutrophication affected its lipid and FA composition. The increase in EPA and DHA proportions could be an adaptive response, which increases stress resistance but could also lead to higher susceptibility to lipid peroxidation TBARS, lipofuscins (20-fold) and GSH concentrations were higher in SMA clams. FA markers indicated terrestrial plant detritus and bacteria are important items in D. chilensis diet. Anthropogenic input in their food could be traced using specific FA as trophic markers.

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.

Chromium- and copper-induced inhibition of photosynthesis in Euglena gracilis analysed on the single-cell level by fluorescence kinetic microscopy.

Here, we investigated effects of copper (Cu) and chromium (Cr) toxicity on two contrasting strains of Euglena gracilis, with and without chloroplasts, grown in culture media promoting either phototrophic or heterotrophic growth. This led to insights into Cr/Cu toxicity mechanisms and into the regulation of phototrophic vs heterotrophic metabolism. Our data strongly suggest that in Cu(2+) and Cr(6+) stressed Euglena photosynthesis is the primary target of damage. In the applied light conditions, this was mainly damage to the photosystem II reaction centre, as shown by single-cell measurements of photochemical fluorescence quenching. Respiration and photosynthetic dark reactions were less sensitive. The malfunctioning photosynthesis enhanced production of reactive oxygen species (mainly superoxide), leading to elevated amounts of carotenoid degradation products. At higher metal concentrations in chloroplast-containing cells, but not white cells, this oxidative stress resulted in increased respiratory oxygen uptake, likely by damage to mitochondria. During growth in nutrient solution promoting heterotrophic metabolism, the cells were able to repair the metal-induced damage to photosynthesis, moderating the inhibition of photochemistry. Growth in medium forcing the cells into photosynthesis increased the investment in photosynthetic pigments. Comparison of the two Euglena strains surprisingly showed that the previously metal-resistant strain lost this resistance during culture.

Gene expression patterns in Euglena gracilis: insights into the cellular response to environmental stress.

To better understand Euglena gracilis gene expression under different stress conditions (Chromium, Streptomycin or darkness), we undertook a survey of the E. gracilis transcriptome by cDNA sequencing and microarray analysis. First, we constructed a non-normalized cDNA library from the E. gracilis UTEX strain and sequenced a total of 1000 cDNAs. Six hundred and ten of these ESTs were similar to either Plantae or Protistae genes (e-value

Oxidative stress in vero cells infected with vesicular stomatitis virus.

Viral-induced apoptosis might be mediated by oxidative stress. It has already been described that cell death in vesicular stomatitis virus (VSV)-infected cells occurs by apoptosis. In this study, oxidative stress parameters present in VSV-infected Vero cells were analyzed. Lipid peroxides (LP) were evaluated in cellular extracts and expressed as thiobarbituric acid-reactive substances. LP levels exhibited a rise at different times post infection, according to the multiplicity of infection (MOI), while the presence of cycloheximide determined a reduction on LP. Also, an increase in protein degradation products and a decrease in polyunsaturated fatty acids content was observed, indicating that cellular proteins and lipids began to be susceptible to degradation during VSV infection. In addition, we analyzed cell viability of VSV-infected Vero cells, which were incubated in the presence of butylated hydroxyanisole. This antioxidant was able to protect Vero cells, at least at MOIs assayed in this study, and to reduce viral yield only when VSV infection was done at MOI 0.05. Further, superoxide dismutases, which occupy the first step within the antioxidant enzyme cascade, also exhibit a rise in VSV-infected Vero cells, at different MOI. These results suggest that both an oxidative stress and an antioxidative cell response precede the induction of apoptosis by VSV.

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.