Effects of Oxygen Availability on Oxidative Stress Biomarkers in the Mediterranean Mussel Mytilus galloprovincialis.

In aquatic environments, hypoxia and oxygen-deficient areas are increasing worldwide. Transitions in oxygen levels can influence the production of reactive oxygen species (ROS), eventually leading to oxidative stress. The transcriptional response of oxidative stress biomarkers was evaluated by qPCR in gill tissue from Mytilus galloprovincialis experimentally subjected to 48-h air exposure followed by 48-h re-oxygenation, as compared to normoxic control mussels. Superoxide dismutases (CuZnsod and Mnsod), catalase (cat), and glutathione S-transferase (gst) were over-expressed early after 8-h air exposure and returned to normoxic levels during re-oxygenation. Moreover, the mRNAs and protein expression patterns of heat shock proteins (HSP70 and HSP90) and metallothioneins (MT-10 and MT-20) were modulated by oxygen availability with increased levels during re-oxygenation suggesting the participation of these cytoprotective mechanisms in the physiological oxidative stress response when oxygen concentration was restored. Overall, the observed modulation of the oxidative stress-related and general stress genes indicates that M. galloprovincialis responds to changes in oxygen availability enhancing the antioxidant potential under low oxygen conditions for dealing with the oxidative burst during future re-oxygenation. The present investigation brings further insights in understanding how intertidal molluscs cope with short-term oxygen variations and gives useful biomarkers for environmental monitoring of hypoxic areas that are predicted to occur in the next future.

Effects of petrochemical contamination on caged marine mussels using a multi-biomarker approach: Histological changes, neurotoxicity and hypoxic stress.

This work was designed to evaluate the biological effects of petrochemical contamination on marine mussels. Mytilus galloprovincialis, widely used as sentinel organisms in biomonitoring studies, were caged at the "Augusta-Melilli-Priolo" industrial site (eastern Sicily, Italy), chosen as one of the largest petrochemical areas in Europe, and Brucoli, chosen as reference site. Chemical analyses of sediments at the polluted site revealed high levels of PAHs and mercury, exceeding the national and international guideline limits. In mussels from the polluted site, severe morphological alterations were observed in gills, mainly involved in nutrient uptake and gas exchange. Changes in serotonergic and cholinergic systems, investigated through immunohistochemical, metabolomics and enzymatic approaches, were highlighted in gills, as well as onset of hypoxic adaptive responses with up-regulation of hypoxia-inducible factor transcript. Overall, the application of a multi-biomarker panel results effective in assessing the biological effects of petrochemical contamination on the health of aquatic organisms.

PCB and OCP accumulation and evidence of hepatic alteration in the Atlantic bluefin tuna, T. thynnus, from the Mediterranean Sea.

Persistent organic pollutants (POPs) are known to act as "obesogens", being fat-soluble and affecting lipid metabolism. The Atlantic bluefin tuna, Thunnus thynnus, are top pelagic predators prone to bioaccumulate and biomagnify environmental contaminants. This study aimed at evaluating POPs-induced ectopic lipid accumulation in liver of adult tuna from the Mediterranean Sea. PCBs and organochlorine pesticides were measured in tuna liver, and marked morphological changes observed, namely poorly compacted tissues, intense vacuolization, erythrocyte infiltration and presence of melanomacrophages. The expression of perilipin, a lipid-droplet marker, positively correlated with the gene expression of PPARγ, a master regulator of adipogenesis, and its heterodimeric partner, RXRα. Changes in metabolites involved in fatty acid biosynthesis and ketogenesis were also observed. Although male bluefin tuna appeared to be more sensitive than females to the adverse effects of environmental obesogens, the alterations observed in tuna liver of both sexes suggest a potential onset of hepatic steatosis.

Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis.

Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally occurring tidal cycles. Mytilus galloprovincialis, being an anoxic/hypoxic-tolerant bivalve, provides an excellent model to investigate the molecular mechanisms regulating oxygen sensing. Across the animal kingdom, inadequacy in oxygen supply is signalled predominantly by hypoxia-inducible factors (HIF) and Hif-prolyl hydroxylases (PHD). In this study, hif-α 5'-end and partial phd mRNA sequences from M. galloprovincialis were obtained. Phylogenetic and molecular characterization of both HIF-α and PHD putative proteins showed shared key features with the respective orthologues from animals strongly suggesting their crucial involvement in the highly conserved oxygen sensing pathway. Both transcripts displayed a tissue-specific distribution with prominent expression in gills. Quantitative gene expression analysis of hif-α and phd mRNAs from gills of M. galloprovincialis demonstrated that both these key sensors are transcriptionally modulated by oxygen availability during the short-time air exposure and subsequent re-oxygenation treatments proving that they are critical players of oxygen-sensing mechanisms in mussels. Remarkably, hif-α gene expression showed a prompt and transient response suggesting the precocious implication of this transcription factor in the early phase of the adaptive response to hypoxia in Mytilus. HIF-α and PHD proteins were modulated in a time-dependent manner with trends comparable to mRNA expression patterns, thus suggesting a central role of their transcriptional regulation in the hypoxia tolerance strategies in marine bivalves. These results provide molecular information about the effects of oxygen deficiency and identify hypoxia-responsive biomarker genes in mussels applicable in ecotoxicological studies of natural marine areas.

Biomarkers of environmental stress in gills of Pinna nobilis (Linnaeus 1758) from Balearic Island.

In aquatic environments, bivalve molluscs are used as sentinel species for environmental biomonitoring. In this study Pinna nobilis specimens, the biggest Mediterranean bivalve, were collected in the Magaluf bay (Mallorca), a touristic location and in a pristine area of the Cabrera National Park as the control location. Histological and histochemical analysis in gills of specimens sampled from Magaluf exhibited evident tissue alterations with high presence of haemocytes. Lower acetylcholinesterase (AChE) activity and protein expression were also found in the gills of specimens collected from Magaluf compared with the control area. The determination of antioxidant enzyme activities, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, showed a higher activities of these antioxidant enzymes and total glutathione content in samples from Magaluf bay than in Cabrera. In conclusion, the present study demonstrated that human activities result in morphological tissue alterations and a reduced AChE activity in gills of P. nobilis. Moreover, these stressful environmental conditions induced an adaptive response in P. nobilis as evidenced by increased antioxidant defences and a decreased AChE activity. CAPSULE: The human activities induce oxidative stress in P. nobilis as evidenced by increased antioxidant defences and a decreased acetylcholinesterase activity.

Developmental abnormalities and neurotoxicological effects of CuO NPs on the black sea urchin Arbacia lixula by embryotoxicity assay.

The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by using 24-well plates. Fertilized eggs were exposed to five doses of CuO NPs ranging from 0.07 to 20 ppb, until pluteus stage. CuO NPs suspensions in artificial seawater formed agglomerates of 80-200 nm size, and copper uptake was 2.5-fold up in larvae exposed to high NP concentrations in respect to control. Developmental delay and morphological alteration, including skeletal abnormalities, were observed, as well as impairment in cholinergic and serotonergic nervous systems. These findings suggest the potential of CuO NPs to interfere with the normal neurotransmission pathways, thus affecting larval morphogenesis. Overall, the embryotoxicity tests are effective for evaluation of nanoparticle effects on the health of aquatic biota. Furthermore, as the black sea urchin A. lixula demonstrated to be vulnerable to NP exposure, it may be a valid bioindicator in marine biomonitoring and ecotoxicological programmes.

Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments.

Multiple anthropogenic activities present along coastal environments may affect the health status of aquatic ecosystems. In this study, specimens of European sea bass (Dicentrarchus labrax) were exposed for 30 days to highly contaminated sediment collected from the industrial area between Augusta and Priolo (Syracuse, Italy), defined as the most mercury polluted site in the Mediterranean. The aim was to evaluate the responses of juvenile D. labrax to highly contaminated sediments, particularly enriched in Hg, in order to enhance the scarce knowledge on the potential compensatory mechanisms developed by organisms under severe stress conditions. Apoptotic and proliferative activities [cell turnover: Proliferating Cell Nuclear Antigen (PCNA) and FAS Ligand (FasL)], onset of hypoxic condition [hypoxia: Hypoxia Inducibile Factor-1α (HIF-1α)], and changes in the neuroendocrine control mechanisms [neurotransmission: Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT), Acetylcholinesterase (AChE), 5-Hydroxytryptamine (5-HT) and 5-Hydroxytryptamine receptor 3 (5-HT3)] were investigated in sea bass gill tissues. In the specimens exposed to the polluted sediment, the occurrence of altered cell turnover may result in impaired gas exchange that leads to a condition of "functional hypoxia". Changes in neurotransmission pathways were also observed, suggesting a remodeling process as an adaptive response to increase the O2-carrying capacity and restore the normal physiological conditions of the gills. Overall, these findings demonstrated that although chronic exposure to heavy metal polluted sediments alters the functioning of both the nervous and endocrine systems, as well as plasticity of the gill epithelium, fish are able to trigger a series of physiological adjustments or adaptations interfering with specific neuroendocrine control mechanisms that enable their long-term survival.

Increased antioxidant response and capability to produce ROS in hemocytes of Pinna nobilis L. exposed to anthropogenic activity.

Environmental pollutants exert immunotoxical effects on aquatic organisms. The aim was to determine the antioxidant response, markers of oxidative damage and reactive oxygen species production in hemocytes of Pinna nobilis, the largest endemic bivalve in the Mediterranean Sea, under anthropogenic pressure. P. nobilis individuals were collected from two locations along Mallorca Island waters attending to different degree of human impact and the hemocytes were obtained. Specimens from the impacted area showed increased activities of the antioxidant enzymes - catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase -, myeloperoxidase activity and reduced glutathione levels. No differences in oxidative damage markers - malondiahdehyde and carbonyl index - were evidenced between the pristine and polluted areas. Hemocytes from the polluted area presented increased capability to generate reactive oxygen species and nitrite/nitrate when activated. In conclusion, the human activities primed hemocytes for oxidative burst and increased the antioxidant mechanism without evidence of oxidative damage.

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis).

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated. The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.

Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax).

Aquatic ecosystems are affected by all the impacts generated by a variety of anthropogenic activities present along coastal environments. The sediment compartment is the final receptor of water-insoluble pollutants, acting both as a sink and as a source of pollutants to the water column, and affecting both nektonic and benthic organisms. The aim of this study is to assess the impact of metals in the sediments collected from two sites in the petrochemical area between Augusta and Priolo (SR, Sicily, Italy) on gills of Dicentrarchus labrax. This was done to enhance the scarce knowledge on the bioavailability of metals bound to sediment and their capacity to interact with the bioindicator species. Various sublethal endpoints were assessed such as histopathological lesions, metallothioneins (MTs) and molecules involved in the homeostasis pathways by immunolocalization and RT-PCR. In the specimens exposed to sediments, the data suggested a reduction of gill cell membrane permeability, which could result in altered osmotic balance and gas exchange. Further, an increase of MT expression was detected, consisted the involvement of this protein in detoxification of toxic non-essential metals. The findings of this study demonstrate that a subchronic test, conducted by using sensitive and sub-lethal endpoints, in combination with chemical analyses, is a powerful tool for early identification of environmental hazards associated with contaminated sediments.