Intertidal limits shape covariation between metabolic plasticity, oxidative stress and telomere dynamics in Pacific oyster (Crassostrea gigas).

In intertidal zones, species such as sessile shellfish exhibit extended phenotypic plasticity to face rapid environmental changes, but whether frequent exposure to intertidal limits of the distribution range impose physiological costs for the animal remains elusive. Here, we explored how phenotypic plasticity varied along foreshore range at multiple organization levels, from molecular to cellular and whole organism acclimatization, in the Pacific oyster (Crassostrea gigas). We exposed 7-month-old individuals for up to 16 months to three foreshore levels covering the vertical range for this species, representing 20, 50 and 80% of the time spent submerged monthly. Individuals at the upper range limit produced energy more efficiently, as seen by steeper metabolic reactive norms and unaltered ATP levels despite reduced mitochondrial density. By spending most of their time emerged, oysters mounted an antioxidant shielding concomitant with lower levels of pro-oxidant proteins and postponed age-related telomere attrition. Instead, individuals exposed at the lower limit range near subtidal conditions showed lower energy efficiencies, greater oxidative stress and shorter telomere length. These results unraveled the extended acclimatization strategies and the physiological costs of living too fast in subtidal conditions for an intertidal species.

Vulnerability of glutathione-depleted Crassostrea gigas oysters to Vibrio species.

Glutathione (GSH) is a major cellular antioxidant molecule participating in several biological processes, including immune function. In this study, we investigated the importance of GSH to oysters Crassostrea gigas immune response. Oysters were treated with the GSH-synthesis inhibitor buthionine sulfoximine (BSO), and the function of immune cells and mortality were evaluated after a bacterial challenge with different Vibrio species. BSO caused a moderate decrease (20-40%) in GSH levels in the gills, digestive gland, and hemocytes. As expected, lower GSH decreased survival to peroxide exposure. Hemocyte function was preserved after BSO treatment, however, oysters became more susceptible to challenges with Vibrio anguillarum, V. alginolyticus, or V. harveyi, but not with V. parahaemolyticus and V. vulnificus, indicating a species-specific vulnerability. Our study indicates that in natural habitats or in mariculture farms, disturbances in GSH metabolism may pre-dispose oysters to bacterial infection, decreasing survival.

Contrasting effects of a classic Nrf2 activator, tert-butylhydroquinone, on the glutathione-related antioxidant defenses in Pacific oysters, Crassostrea gigas.

Nrf2 is a well-known transcription factor controlling a number of antioxidant defense-related genes, which is understudied in bivalves. In this study, oysters Crassostrea gigas were exposed for 24, 48 and 96 h to 10 or 30 µM tert-butylhydroquinone (tBHQ), a classic Nrf2 activator. At 96 h, a clear induction of GSH-related antioxidant defenses was observed in gills of tBHQ-exposed animals, including GSH, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR). Unexpectedly, the activities of GST, GPx and GR were significantly decreased 24 h after tBHQ treatment, suggesting a possible inhibition, which was supported by in vitro experiments. GR mRNA (24 h) and protein levels (24 and 96 h) were increased by tBHQ treatment, confirming its induction, possibly by the Nrf2 pathway. The conserved domains at C. gigas Keap1 and Nrf2 proteins and the clear induction of GSH-related antioxidant defenses by tBHQ, a classical Nrf2 inducer, support the idea of a functional Nrf2/Keap1 pathway in bivalves. tBHQ also proved to be a tool to explore redox regulatory mechanisms in bivalves.

Hypoxia effects on oxidative stress and immunocompetence biomarkers in the mussel Perna perna (Mytilidae, Bivalvia).

This study investigated the effects of hypoxia on oxidative stress response and immune function in mussels Perna perna exposed to air for 6, 12, 24 and 48 h. In air-exposed mussels, the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione reductase (GR) activities were lower in gill tissues (24-48 h) and digestive gland (12 h), while the glutathione peroxidase and GR activities were increased in the digestive gland (48 h). In both tissues, aerial exposure promoted a rapid (6 h) and persistent (up to 48 h) increase of glutathione levels. Decreased hemocyte count and viability, as well as increased phagocytic activity and cellular adhesion capacity were detected after prolonged aerial exposure (>12 h). In summary, induction of thiol pools, altered antioxidant enzyme activities, and activation of immune responses were detected in hypoxia exposed brown mussels, indicating hypoxia induced tissue-specific responses in both antioxidant and immune systems.

Upregulating Nrf2-dependent antioxidant defenses in Pacific oysters Crassostrea gigas: Investigating the Nrf2/Keap1 pathway in bivalves.

Analysis of the Pacific oyster Crassostrea gigas annotated genome revealed genes with conserved sequences belonging to typical cap 'n' collar Nrf2 domain, a major player in antioxidant protection, and domains belonging to Nrf2 cytoplasmic repressor (Keap1), but little is known about Nrf2/Keap1 induction in bivalves. C. gigas were exposed to waterborne 10 and 30µM curcumin, a known inducer of the mammalian Nrf2. Curcumin disappeared from the seawater after 10h, and accumulated in the gills (10h) and digestive gland (10-96h). A clear induction of glutathione (GSH)-related antioxidant defenses was observed at 96h in the gills of curcumin exposed animals (10 and 30µM), including GSH levels, and the activity of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). This response was completely absent in the digestive gland, in line with the idea that bivalve gills act as a major site for antioxidant protection under acute exposure. The relative mRNA levels coding glutamate-cysteine ligase, GR, GPx2 and GSTpi were clearly induced by curcumin treatment (30µM, 24h). Curcumin pre-treatment for 96h increased oyster resistance to cumene hydroperoxide, but neither Nrf2 nor Keap1 genes were modulated by curcumin. However, the conserved sequences belonging to typical Nrf2 and Keap1 domains, and the notorious induction of antioxidant defense-related genes known to be controlled by Nrf2 in mammals, indicates a functional Nrf2/Keap1 pathway in bivalves, and curcumin seems to be a new tool to investigate the antioxidant response in bivalves.

Modulation of Brain Glutathione Reductase and Peroxiredoxin 2 by α-Tocopheryl Phosphate.

α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound.

How important are glutathione and thiol reductases to oyster hemocyte function?

Bivalves are animals with worldwide distribution. Although they play key roles in economic activities, human feeding and environmental studies, there is a considerable lack of knowledge about the relationship between their immune system and antioxidant defenses. Here, we performed an in vitro experiment where Crassostrea gigas hemocytes were exposed to the electrophilic compound 1-chloro-2,4-dinitrobenzene (CDNB, 0.1-50 µM) for one hour. CDNB treatment clearly disturbed thiol homeostasis, causing a concentration-dependent decrease in the glutathione (GSH) content and a decrease in the activity of two thiol reductases, glutathione reductase (GR - 2.5 and 50 µM CDNB) and thioredoxin reductase (TrxR - only 50 µM CDNB). The MTT reduction assay showed that none of the CDNB concentrations tested significantly altered cell viability. However, there was a decrease in the hemocyte's ability to uptake the neutral red dye, which indicates lysosomal impairment (≥12.5 µM CDNB). Cellular immunocompetence was further investigated and, despite the lower GSH content, GR activity and impairment in lysosome integrity, hemocyte functions (adhesion capacity, phagocytosis of latex beads and laminarin-induced ROS production) were preserved in the 2.5 and 12.5 µM CDNB treatments. These results suggest a minor importance of thiol pools and GR activity in C. gigas hemocyte's immunocompetence, in an in vitro acute exposure model. The 50 µM CDNB treatment, however, significantly compromised all the measured hemocyte functions. This response was associated with TrxR inhibition, increased lysosome impairment, decreased GSH content, and lower GR activity. Therefore, it seems that TrxR may be particularly important for the hemocyte function, or, alternatively, it is only affected when a deeply aggravated scenario in thiol homeostasis is set up. Such findings point out the need for further studies towards a better understanding of antioxidant and immune defenses interactions in bivalve cellular systems.

The biological importance of glutathione peroxidase and peroxiredoxin backup systems in bivalves during peroxide exposure.

Organic peroxide elimination in eukaryotes essentially depends on glutathione peroxidase (GPx) and peroxiredoxin (Prx) enzymes, which are supported by their respective electron donors, glutathione (GSH) and thioredoxin (Trx). This system depends on the ancillary enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) to maintain GSH and Trx in their reduced state. This study discusses the biological importance of GR and TrxR in supporting GPx and Prx during cumene hydroperoxide (CHP) exposure in brown mussel Perna perna. ZnCl2 or 1-chloro-2,4-dinitrobenze (CDNB) was used to decrease GR and TrxR activities in gills, as already reported with mammals and bivalves. ZnCl2 exposure lowered GR activity (28%), impaired the in vivo CHP decomposition and decreased the survival rates under CHP exposure. CDNB decreased GR (54%) and TrxR (73%) activities and induced glutathione depletion (99%), promoting diminished peroxide elimination and survival rates at a greater extent than ZnCl2. CDNB also increased the susceptibility of hemocytes to CHP toxicity. Despite being toxic and causing mortality at longer exposures, short (2 h) exposure to CHP promoted an up regulation of GSH (50 and 100 µM CHP) and protein-thiol (100 µM CHP) levels, which was blocked by ZnCl2 or CDNB pre-exposure. Results highlight the biological importance of GSH, GR and TrxR in supporting GPx and Prx activities, contributing to organic peroxides elimination and mussel survival under oxidative challenges. To our knowledge, this is the first work that demonstrates, albeit indirectly, the biological importance of GPx/GR/GSH and Prx/TrxR/Trx systems on in vivo organic peroxide elimination in bivalves.

Antioxidant deficit in gills of Pacific oyster (Crassostrea gigas) exposed to chlorodinitrobenzene increases menadione toxicity.

Disturbances in antioxidant defenses decrease cellular protection against oxidative stress and jeopardize cellular homeostasis. To knock down the antioxidant defenses of Pacific oyster Crassostrea gigas, animals were pre-treated with 1-chloro-2,4-dinitrobenzene (CDNB) and further challenged with pro-oxidant menadione (MEN). CDNB pre-treatment (10 µM for 18 h) was able to consume cellular thiols in gills, decreasing GSH (53%) and decrease protein thiols (25%). CDNB pre-treatment also disrupted glutathione reductase and thioredoxin reductase activity in the gills, but likewise strongly induced glutathione S-transferase activity (270% increase). Surprisingly, hemocyte viability was greatly affected 24 h after CDNB removal, indicating a possible vulnerability of the oyster immune system to electrophilic attack. New in vivo approaches were established, allowing the identification of higher rates of GSH-CDNB conjugate export to the seawater and enabling the measurement of the organic peroxide consumption rate. CDNB-induced impairment in antioxidant defenses decreased the peroxide removal rate from seawater. After showing that CDNB decreased gill antioxidant defenses and increased DNA damage in hemocytes, oysters were further challenged with 1 mM MEN over 24 h. MEN treatment did not affect thiol homeostasis in gills, while CDNB pre-treated animals recovered GSH and PSH to the control level after 24 h of depuration. Interestingly, MEN intensified GSH and PSH loss and mortality in CDNB-pre-treated animals, showing a clear synergistic effect. The superoxide-generating one-electron reduction of MEN was predominant in gills and may have contributed to MEN toxicity. These results support the idea that antioxidant-depleted animals are more susceptible to oxidative attack, which can compromise survival. Data also corroborate the idea that gills are an important detoxifying organ, able to dispose of organic peroxides, induce phase II enzymes, and efficiently export GSH-CDNB conjugates.

Selenium in water enhances antioxidant defenses and protects against copper-induced DNA damage in the blue mussel Mytilus edulis.

Selenium and copper are naturally occurring elements in the environment that have important roles in cellular function. Selenium is known for its role in antioxidant defense, whereas copper is a redox-active metal capable of acting as a pro-oxidant. We investigated the effects of short term selenium (Na(2)SeO(3)) supplementation (4 µg/L for 3 days) on antioxidant parameters of the blue mussel, Mytilus edulis, and its possible protective effects against a subsequent copper (CuSO(4)) exposure (56 µg/L for 3 days). Selenium supplementation caused a 4-fold increase in glutathione levels in gills. The activity of selenium-dependent glutathione peroxidase was modulated by selenium in gills (2-fold increase) and also in cell-free haemolymph (40% increase). Copper exposure produced decreases in protein thiol levels (35%) and in thioredoxin reductase activity (60%) in gills and induced an increase in DNA damage in haemocytes (70% increase in % tail DNA observed using the comet assay). The decrease in thioredoxin reductase activity may constitute a mechanism of copper toxicity in bivalves, warranting further investigation. Pre-treatment with selenium largely prevented these deleterious effects of copper on protein thiols, thioredoxin reductase activity and DNA damage. The results suggest that induction of key antioxidant defenses such as glutathione and selenium-dependent glutathione peroxidase, as a result of selenium supplementation, may play an important role in protection of aquatic organisms against oxidative stress.

Comparative study of various immune parameters in three bivalve species during a natural bloom of Dinophysis acuminata in Santa Catarina Island, Brazil.

This study aimed to verify if Dinophysis acuminata natural blooms affected the immune system of three bivalves: the oyster, Crassostrea gigas, the mussel, Perna perna, and the clam, Anomalocardia brasiliana. Animals were obtained from a renowned mariculture farm in the southern bay of Santa Catarina Island during, and 30 days after (controls), an algal bloom. Various immunological parameters were assessed in the hemolymph of the animals: total and differential hemocyte counts, percentage of apoptotic hemocytes, protein concentration, hemagglutinating titer and phenoloxidase activity. The results showed that the mussel was the most affected species, with several altered immune parameters, whereas the immunological profile of clams and oysters was partially and completely unaffected, respectively.