Time- and dose-dependent biological effects of a sub-chronic exposure to realistic doses of salicylic acid in the gills of mussel Mytilus galloprovincialis.

Among nonsteroidal anti-inflammatory drugs (NSAIDs) commonly found in seawater and wastewater, salicylic acid (SA) represents one of the most persistent and hazardous compounds for aquatic organisms. This study was therefore designed to elucidate the biological effects of SA in mussel Mytilus galloprovincialis. During a sub-chronic exposure (12 days), mussels were exposed to five realistic concentrations of SA (C1: 0.05 µg/L; C2: 0.5 µg/L; C3: 5 µg/L; C4: 50 µg/L; C5: 100 µg/L) and gills, selected as the target organ, were collected at different time points (T3: 3 days; T5: 5 days; T12: 12 days). Exposure to SA induced no histological alterations in mussel gills, despite a relevant hemocyte infiltration was observed throughout the exposure as a defensive response to SA. Temporal modulation of glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) activities suggested the occurrence of antioxidant and detoxifying responses against SA exposure, while lipid peroxidation (LPO), except for a partial increase at T3, was prevented. Inhibition of the cholinergic system was also reported by reduced acetylcholinesterase (AChE) activity, mainly at T12. Overall, findings from this study contribute to enlarge the current knowledge on the cytotoxicity of SA, on non-target aquatic organisms, and might for the enhancement of new ecopharmacovigilance programs and optimization of the efficacy of wastewater treatment plants for mitigation of pharmaceutical pollution in coastal areas.

Assessment of the effectiveness of a novel BioFilm-Membrane BioReactor oil-polluted wastewater treatment technology by applying biomarkers in the mussel Mytilus galloprovincialis.

Petrochemical industries and oil refineries are sources of hazardous chemicals into the aquatic environments, and often a leading cause of reduced oxygen availability, thus resulting in adverse effects in biota. This study is an expansion of our previous work on the assessment of the BioFilm-Membrane Bioreactor (BF-MBR) to mitigate the impact of oil-polluted wastewater on marine environments. Specifically, this study evaluated the reduction of selected chemical constituents (hydrocarbons and trace metals) and toxicity related to hypoxia and DNA damage to mussels Mytilus galloprovincialis, before and after treatment of oil-polluted wastewater with the BF-MBR. The application of a multidisciplinary approach provided evidence of the efficiency of BF-MBR to significantly reducing the pollutants load from oily contaminated seawaters. As result, the health status of mussels was preserved by a hypoxic condition due to oily pollutants, as evidenced by the modulation in the gene expression of HIF-1α and PHD and changes in the level of hypotaurine and taurine. Moreover, ameliorative effects in the energy metabolism were also found in mussel gills showing increased levels of glycogen, glucose and ATP, as well as a mitigated genotoxicity was revealed by the Micronucleus and Comet assays. Overall, findings from this study support the use of the BF-MBR as a promising treatment biotechnology to avoid or limiting the compromise of marine environments from oil pollution.

Embryotoxicity of polystyrene microplastics in zebrafish Daniorerio.

In the recent years, increasing scientific and societal concern has been raised over the presence and accumulation of plastic debris in the environment and the effects of microplastics (MPs) that can easily interact with biota. In order to elucidate the impact of MPs at the critical development stages of freshwater fish species, a fish embryo toxicity test was herein performed on the zebrafish Danio rerio, exposed to 10 µm polystyrene MPs at 200 particles/mL for 120 hpf. After exposure, accumulation of MPs in larvae was measured, survival, hatching and larvae development were monitored and the oxidant/anti-oxidant responses and cellular detoxification evaluated. No impact on survival of developing zebrafish was revealed, but a moderate delay in hatching was observed. Alterations in larvae development were recorded with zebrafish exhibiting serious deformities, mainly at the level of column and tail, as well as a compromised integrity of the visual structure of the eyes. Moreover, increased levels of gene transcription involved in the oxidative stress (sod1, sod2 and cat) and in cellular detoxification (gst and cyp) were also detected in MPs-exposed zebrafish larvae. Overall, this research work provides new insights on the ecotoxicological impact of polystyrene MPs on the critical developmental stages of a freshwater fish species, therefore enhancing the current knowledge of the environmental risk posed by MPs to the aquatic ecosystem.

Time-dependent metabolic disorders induced by short-term exposure to polystyrene microplastics in the Mediterranean mussel Mytilus galloprovincialis.

In the modern society, plastic has achieved a crucial status in a myriad of applications because of its favourable properties. Despite the societal benefits, plastic has become a growing global concern due to it is persistence and bioavailability as microplastics (MPs) to aquatic biota. In order to provide mechanistic insights into the early toxicity effects of MPs on aquatic invertebrates, a short-term (up to 72 h) exposure to 3 µm red polystyrene MPs (50 particles/mL) was conducted on marine mussels Mytilus galloprovincialis, selected as model organism for their ability to ingest MPs and their commercial relevance. The use of protonic Nuclear Magnetic Resonance (1H NMR)-based metabolomics, combined with chemometrics, enabled a comprehensive exploration at fixed exposure time-points (T24, T48, T72) of the impact of MPs accumulated in mussel digestive glands, chosen as the major site for pollutants storage and detoxification processes. In detail, 1H NMR metabolic fingerprints of MP-treated mussels were clearly separated from control and grouped for experimental time-points by a Principal Component Analysis (PCA). Numerous metabolites, including amino acids, osmolytes, metabolites involved in energy metabolism, and antioxidants, participating in various metabolic pathways significantly changed over time in MP-exposed mussel digestive glands related to control, reflecting also the fluctuations in MPs accumulation and pointing out the occurrence of disorders in amino acid metabolism, osmotic equilibrium, antioxidant defense system and energy metabolism. Overall, the present work provides the first insights into the early mechanisms of toxicity of polystyrene MPs in marine invertebrates.

Waste Valorization via Hermetia Illucens to Produce Protein-Rich Biomass for Feed: Insight into the Critical Nutrient Taurine.

Insects have been recognized as sustainable alternative sources of nutrients for food and feed. The Black Soldier Fly (BSF), Hermetia illucens, is a particularly promising species for its great potential in the waste valorization to produce, during the bioconversion process, high-value fat and proteins that currently represent a valuable source for fish feed. The present study aims to evaluate the efficiency to use substrate proteins in two different BSF developmental stages as sustainable biotechnological tools for vegetable waste management. We provide insights into the nutritional values of both V instar larvae and prepupae in terms of valuable amino acids with special focus on taurine, a crucial nutrient for fish. Moreover, we cloned four key genes from BSF involved in the taurine biosynthesis pathway, 2-aminoethanethiol dioxygenase (Hiado), cysteine dioxygenase (Hicdo), cysteine sulfonate decarboxylase (Hicsad), and glutamate decarboxylase (Higad). The gene expression analysis in larvae and prepupae by qPCR showed development-specific profiles suggesting they influence the taurine content during BSF development. These findings showed peculiar phenotypes in larvae and prepupae that can be selected for different biotechnological applications as sustainable source of relevant amino acids and taurine to support the increasing demand for animal feed and aquafeed in the next decades.

Hermetia illucens (Diptera: Stratiomydae) larvae and prepupae: Biomass production, fatty acid profile and expression of key genes involved in lipid metabolism.

The Black Soldier Fly (BSF) Hermetia illucens provides a promising strategy in the waste valorisation process and a sustainable alternative source of valuable nutrients, including lipids for food and feed. In the present study, the differences in growth performances and nutritional values of BSF V instar larvae and prepupae reared on vegetable waste were analyzed and compared focusing on fat content. V instar larvae showed higher capacity to bioconvert the substrate into biomass than prepupae. The nutritional composition and the fatty acid profiles were dependent on the developmental stage. The expression levels of acetyl-CoA carboxylase (acc), fatty acid synthase (fas), lipase (lip) and acyl-CoA dehydrogenase (acd) genes involved in the lipid metabolism pathway and herein characterized for the first time, were evaluated in order to understand the molecular basis underlying the observed differences in fatty acid profiles. Our results suggest that the different fatty acid profiles of BSF V instar larvae and prepupae may be related to the modulation of the lipid metabolism-related genes expression during larval development. Our study highlights substantial differences between H. illucens V instar larvae and prepupae giving important features regarding the opportunity to modulate the preferable fatty acid profile to meet the industrial requirements.

Responses of marine mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) after infection with the pathogen Vibrio splendidus.

Bivalve molluscs possess effective cellular and humoral defence mechanisms against bacterial infection. Although the immune responses of mussels to challenge with pathogenic vibrios have been largely investigated, the effects at the site of injection at the tissue level have not been so far evaluated. To this aim, mussels Mytilus galloprovincialis were herein in vivo challenged with Vibrio splendidus to assess the responses induced in hemolymph and posterior adductor muscle (PAM), being the site of bacterial infection. The number of living intra-hemocyte bacteria increased after the first hour post-injection (p.i.), suggesting the occurrence of an intense phagocytosis, while clearance was observed within 24 h p.i. A recruitment of hemocytes at the injection site was found in mussel PAM, together with marked morphological changes in the volume of muscular fibers, with a recovery of muscle tissue organization after 48 h p.i. A concomitant impairment in the osmoregulatory processes were observed in PAM by an initial inhibition of aquaporins and increased immunopositivity of Na+/K+ ATPase ionic pump, strictly related to the histological alterations and hemocyte infiltration detected in PAM. Accordingly, an intense cell turnover activity was also recorded following the infection event. Overall, results indicated the hemolymph as the system responsible for the physiological adaptations in mussels to stressful factors, such as pathogenicity, for the maintenance of homeostasis and immune defence. Also, the osmotic balance and cell turnover can be used as objective diagnostic criteria to evaluate the physiological state of mussels following bacterial infection, which may be relevant in aquaculture and biomonitoring studies.

Copper oxide nanoparticles induce the transcriptional modulation of oxidative stress-related genes in Arbacia lixula embryos.

Copper oxide nanoparticles (CuO NPs) are widely used in various industrial applications, i.e. semiconductor devices, batteries, solar energy converter, gas sensor, microelectronics, heat transfer fluids, and have been recently recognized as emerging pollutants of increasing concern for human and marine environmental health. Therefore, the toxicity of CuO NPs needs to be thoroughly understood. In this study, we evaluated the potential role of oxidative stress in CuO NP toxicity by exploring the molecular response of Arbacia lixula embryos to three CuO NP concentrations (0.7, 10, 20 ppb) by investigating the transcriptional patterns of oxidative stress-related genes (catalase and superoxide dismutase) and metallothionein, here cloned and characterized for the first time. Time- and concentration-dependent changes in gene expression were detected in A. lixula embryos exposed to CuO NPs, up to pluteus stage (72 h post-fertilization, hpf), indicating that oxidative stress is one of the toxicity mechanisms for CuO NPs. These findings provide new insights into the comprehension of the molecular mechanisms underlying copper nanoparticle toxicity in A. lixula sea urchin and give new tools for monitoring of aquatic areas, thus corroborating the suitability of this embryotoxicity assay for future evaluation of impacted sites.

Baseline levels of metabolites in different tissues of mussel Mytilus galloprovincialis (Bivalvia: Mytilidae).

The Mediterranean mussel Mytilus galloprovincialis (Lamarck 1819) is a popular shellfish commonly included in human diet and is routinely used as bioindicator in environmental monitoring programmes worldwide. Recently, metabolomics has emerged as a powerful tool both in food research and ecotoxicology for monitoring mussels' freshness and assessing the effects of environmental changes. However, there is still a paucity of data on complete metabolic baseline of mussel tissues. To mitigate this knowledge gap, similarities and differences in metabolite profile of digestive gland (DG), gills (G), and posterior adductor muscle (PAM) of aquaculture-farmed M. galloprovincialis were herein investigated by a proton nuclear magnetic resonance (1H NMR)-based metabolomic approach and discussed considering their physiological role. A total of 44 metabolites were identified in mussel tissues and grouped in amino acids, energy metabolites, osmolytes, neurotransmitters, nucleotides, alkaloids, and miscellaneous metabolites. A PCA showed that mussel tissues clustered separately from each other, suggesting a clear differentiation in their metabolic profiles. A Venn diagram revealed that mussel DG, G and PAM shared 27 (61.36%) common metabolites, though with different concentrations. Osmolytes were found to dominate the metabolome of all tissues. The DG exhibited higher level of glutathione and carbohydrates. The G showed greater level of osmolytes and the exclusive presence of neurotransmitters, namely acetylcholine and serotonin. In PAM higher levels of energetics-related metabolites were found. Overall, findings from this study are helpful for a better understanding of mussel tissue-specific physiological functions as well as for future NMR-based metabolomic investigations of marine mussel health and safety.

Food safety using NMR-based metabolomics: Assessment of the Atlantic bluefin tuna, Thunnus thynnus, from the Mediterranean Sea.

Seafood is a valuable component of human diet because of its nutritional properties. The Atlantic bluefin tuna Thunnus thynnus is a scombroid fish highly appreciated as seafood worldwide. Being a top pelagic predator, concerns have been raised over human health risks due to its consumption. Therefore, herein it was comprehensively evaluated the health status and potential metabolite shifts between sexes of bluefin tuna from the Mediterranean Sea, proved to bioaccumulate environmental obesogens, namely polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) at hepatic level. To this aim, nuclear magnetic resonance (NMR)-based metabolomics was applied on liver of post-spawning tuna. Findings from this study pointed out that hepatic bioaccumulation of PCBs and OCPs induced differential metabolic disturbances between sexes, attributable to the reproductive stage at which tuna were caught. Alterations were mainly found in energy-producing metabolic pathways (with changes in acetate, acetoacetate, malonate, and lactate), amino acid (with changes in BCAA, alanine, sarcosine, and tyrosine), and lipid (with changes in choline and phosphocholine) metabolism, with activation of fatty acids biosynthesis and ketogenesis in male tuna. Overall, according to the current European legislation on maximum levels of contaminants in seafood, the consumption of bluefin tuna does not represent a risk for human health.

Molecular characterization and expression analysis of heat shock protein 70 and 90 from Hermetia illucens reared in a food waste bioconversion pilot plant.

Two full-length cDNAs of heat shock protein (HSP) genes (Hihsp70 and Hihsp90) were cloned from the black soldier fly (BSF) Hermetia illucens larvae reared in a food waste bioconversion pilot plant. The Hihsp70 and Hihsp90 transcripts were 2243 and 2507bp long, contained 1923 and 2166bp open reading frames encoding proteins of 640 and 721 amino acids with a molecular mass of 69.8 and 83kDa, respectively. Comparative analysis of protein sequences revealed the presence of the conserved HSP motifs in both proteins, showing high homology to their counterparts in other insect species from six different orders. Hihsp70 and Hihsp90 transcriptional expression profiles during two key developmental stages in the bioconversion process were evaluated by quantitative real time PCR showing that both genes were modulated during larval development. HiHsp70 mRNA expression levels during the II instar larvae was higher in respect to the V instar larvae. A similar difference in mRNA expression levels, but in a less extent, was found for the Hihsp90. Moreover, a diverse transcript level between the two genes at the V larval stage was observed where Hihsp90 was up-regulated compared to Hihsp70. These results suggested the involvement of Hsp70 and Hsp90 in H. illucens development and provide further evidences on the ecological and evolutionary importance of HSPs in the insect developmental processes together with valuable information on molecular features of adaptability to peculiar rearing conditions during food waste bioconversion.

Sex steroids and metabolic responses in mussels Mytilus galloprovincialis exposed to drospirenone.

Drospirenone (DRO) is a synthetic progestin derived from 17α-spironolactone with a pharmacological mechanism of action similar to progesterone. Despite its wide use as pharmaceutical and consequent continuous release into the aquatic environment, DRO effects have been poorly investigated on aquatic biota. In order to unravel the toxicity mechanisms of DRO, mussels Mytilus galloprovincialis were exposed for 7 days to different concentrations of DRO, namely 20ng/L (Low; L), 200ng/L (Medium; M), 2000ng/L (High; H) and 10µg/L (Super High; SH) nominal doses. Following exposure, no significant effect was observed on gonad maturation of treated and untreated mussels. The levels of progesterone (P4) and testosterone (T) were measured in mantle/gonad tissues and no significant alteration detected after exposure. However, the application of a protonic nuclear magnetic resonance (1H NMR)-based metabolomics approach enabled a comprehensive assessment of DRO effects in mussels. Specifically, 1H NMR metabolic fingerprints of digestive glands of DRO treated mussel groups were clearly separated from each other and from controls through a principal component analysis (PCA). Moreover, a number of metabolites involved in different metabolic pathways were found to significantly change in DRO-exposed mussels compared to control, suggesting the occurrence of alterations in energy metabolism, amino acids metabolism, and glycerophospholipid metabolism. Overall, despite no changes in gonad maturation and steroids levels were recorded in mussels after DRO exposure, the metabolomics approach demonstrated its effectiveness and high sensitivity in elucidating DRO-induced metabolic disturbances in marine mussels, and thus its usefulness in the environmental risk assessment of pharmaceuticals.

1H NMR-based metabolomics investigation on the effects of petrochemical contamination in posterior adductor muscles of caged mussel Mytilus galloprovincialis.

Environmental metabolomics is a high-throughout approach that provides a snapshot of the metabolic status of an organism. In order to elucidate the biological effects of petrochemical contamination on aquatic invertebrates, mussels Mytilus galloprovincialis were caged at the "Augusta-Melilli-Priolo" petrochemical area and Brucoli (Sicily, south Italy), chosen as the reference site. After confirming the elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and mercury (Hg) in Augusta sediments in our previous work (Maisano et al., 2016a), herein an environmental metabolomics approach based on protonic nuclear magnetic resonance (1H NMR), coupled with chemometrics, was applied on the mussel posterior adductor muscle (PAM), the main muscular system in bivalve molluscs. Amino acids, osmolytes, energy storage compounds, tricarboxylic acid cycle intermediates, and nucleotides, were found in PAM NMR spectra. Principal Component Analysis (PCA) indicated that mussels caged at the polluted site clustered separately from mussels from the control area, suggesting a clear differentiation between their metabolic profiles. Specifically, disorders in energy metabolism, alterations in amino acids metabolism, and disturbance in the osmoregulatory processes were observed in mussel PAM. Overall, findings from this work demonstrated the usefulness of applying an active biomonitoring strategy for environmental risk assessment, and the effectiveness of metabolomics in elucidating changes in metabolic pathways of aquatic organisms caged at sites differentially contaminated, and thus its suitability to be applied in ecotoxicological studies.

Alteration of neurotransmission and skeletogenesis in sea urchin Arbacia lixula embryos exposed to copper oxide nanoparticles.

The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula, an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N-acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130, did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota.

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.

Advances in understanding the mechanisms of mercury toxicity in wild golden grey mullet (Liza aurata) by 1H NMR-based metabolomics.

Mercury (Hg) is recognized as a dangerous contaminant due to its bioaccumulation and biomagnification within trophic levels, leading to serious health risks to aquatic biota. Therefore, there is an urgent need to unravel the mechanisms underlying the toxicity of Hg. To this aim, a metabolomics approach based on protonic nuclear magnetic resonance (1H NMR), coupled with chemometrics, was performed on the gills of wild golden grey mullets L. aurata living in an Hg-polluted area in Ria de Aveiro (Portugal). Gills were selected as target organ due to their direct and continuous interaction with the surrounding environment. As a consequence of accumulated inorganic Hg and methylmercury, severe changes in the gill metabolome were observed, indicating a compromised health status of mullets. Numerous metabolites, i.e. amino acids, osmolytes, carbohydrates, and nucleotides, were identified as potential biomarkers of Hg toxicity in fish gills. Specifically, decrease of taurine and glycerophosphocholine, along with increased creatine level, suggested Hg interference with the ion-osmoregulatory processes. The rise of lactate indicated anaerobic metabolism enhancement. Moreover, the increased levels of amino acids suggested the occurrence of protein catabolism, further supported by the augmented alanine, involved in nitrogenous waste excretion. Increased level of isobutyrate, a marker of anoxia, was suggestive of onset of hypoxic stress at the Hg contaminated site. Moreover, the concomitant reduction in glycerophosphocholine and phosphocholine reflected the occurrence of membrane repair processes. Finally, perturbation in antioxidant defence system was revealed by the depletion in glutathione and its constituent amino acids. All these data were also compared to the differential Hg-induced metabolic responses previously observed in liver of the same mullets (Brandão et al., 2015). Overall, the environmental metabolomics approach demonstrated its effectiveness in the evaluation of Hg toxicity mechanisms in wild fish under realistic environmental conditions, uncovering tissue-specificities regarding Hg toxic effects namely in gills and liver.

Free fatty acid profiling of marine sentinels by nanoLC-EI-MS for the assessment of environmental pollution effects.

The present work aims to elucidate the free fatty acid (FFA) profile of the mussel Mytilus galloprovincialis caged in an anthropogenically impacted area and in a reference site through an innovative and validated analytical approach for the assessment of biological alterations induced by marine pollution. The FFA pattern is involved in the regulation of different cellular pathways and differs with respect to metabolic stimuli. To this purpose, the lipid fraction of mussels coming from both sampling areas was extracted and the FFA fractions were isolated and purified by a solid phase extraction; then, nano-scale liquid chromatography coupled to electron ionization mass spectrometry (nanoLC-EI-MS) was employed for the characterization of the two samples. A total of 19 and 17 FFAs were reliably identified in the mussels coming from the reference and polluted site, respectively. Significant qualitative and quantitative differences found in saturated, monounsaturated and polyunsaturated species may be exploited as typical pollution biomarkers (e.g. alteration of the fatty acid biosynthetic system and lipotoxicity) and explain adverse and compromising effects (e.g. oxidative stress and inflammatory processes) related to environmental pollution.

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.

Effects of titanium dioxide nanoparticle exposure in Mytilus galloprovincialis gills and digestive gland.

Despite the wide use of nanoscale materials in several fields, some aspects of the nanoparticle behavior have to be still investigated. In this work, we faced the aspect of environmental effects of increasing concentrations of TiO2NPs using the Mytilus galloprovincialis as an animal model and carrying out a multidisciplinary approach to better explain the results. Bioaccumulation suggested that the gills and digestive gland are the most sensitive organs to TiO2NP exposure. Histological observations have evidenced an altered tissue organization and a consistent infiltration of hemocytes, as a consequence of the immune system activation, even though an increase in lipid peroxidation is uncertain and DNA damage became relevant only at high exposure dose (10 mg/L) or for longer exposure time (96 h). However, the over expression of SOD1 mRNA strengthen the concept that the toxicity of TiO2NPs could occur indirectly by ROS production. TEM analysis showed the presence of multilamellar bodies, RER fragmentation, and cytoplasmic vacuolization within relevant presence of dense granules, residual bodies, and lipid inclusions. These findings support the evidence of an initial inflammatory response by the cells of the target organs leading to apoptosis. In conclusion, we can state that certainly the exposure to TiO2NPs has affected our animal model from cellular to molecular levels. Interestingly, the same responses are caused by lower TiO2NP concentration and longer exposure time as well as higher doses and shorter exposure. We do not know if some of the conditions detected are reversible, then further studies are required to clarify this aspect.

Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining (1)H NMR metabolomics and conventional biochemical assays.

Oxidative stress has been described as a key pathway to initiate mercury (Hg) toxicity in fish. However, the mechanisms underlying Hg-induced oxidative stress in fish still need to be clarified. To this aim, environmental metabolomics in combination with a battery of conventional oxidative stress biomarkers were applied to the gills of golden grey mullet (Liza aurata) collected from Largo do Laranjo (LAR), a confined Hg contaminated area, and São Jacinto (SJ), selected as reference site (Aveiro Lagoon, Portugal). Higher accumulation of inorganic Hg and methylmercury was found in gills of fish from LAR relative to SJ. Nuclear magnetic resonance (NMR)-based metabolomics revealed changes in metabolites related to antioxidant protection, namely depletion of reduced glutathione (GSH) and its constituent amino acids, glutamate and glycine. The interference of Hg with the antioxidant protection of gills was corroborated through oxidative stress endpoints, namely the depletion of glutathione peroxidase and superoxide dismutase activities at LAR. The increase of total glutathione content (reduced glutathione+oxidized glutathione) at LAR, in parallel with GSH depletion aforementioned, indicates the occurrence of massive GSH oxidation under Hg stress, and an inability to carry out its regeneration (glutathione reductase activity was unaltered) or de novo synthesis. Nevertheless, the results suggest the occurrence of alternative mechanisms for preventing lipid peroxidative damage, which may be associated with the enhancement of membrane stabilization/repair processes resulting from depletion in the precursors of phosphatidylcholine (phosphocholine and glycerophosphocholine), as highlighted by NMR spectroscopy. However, the observed decrease in taurine may be attributable to alterations in the structure of cell membranes or interference in osmoregulatory processes. Overall, the novel concurrent use of metabolomics and conventional oxidative stress endpoints demonstrated to be sensitive and effective towards a mechanistically based assessment of Hg toxicity in gills of wild fish, providing new insights into the toxicological pathways underlying the oxidative stress.