Long-term exposure of Mytilus galloprovincialis to diclofenac, Ibuprofen and Ketoprofen: Insights into bioavailability, biomarkers and transcriptomic changes.

Non-steroidal anti-inflammatory drugs (NSAIDs) represent a growing concern for marine ecosystems due to their ubiquitous occurrence and documented adverse effects on non-target organisms. Despite the remarkable efforts to elucidate bioaccumulation and ecotoxicological potential under short-term conditions, limited and fragmentary information is available for chronic exposures. In this study bioavailability, molecular and cellular effects of diclofenac (DIC), ibuprofen (IBU) and ketoprofen (KET) were investigated in mussels Mytilus galloprovincialis exposed to the realistic environmental concentration of 2.5 µg/L for up to 60 days. Results indicated a significant accumulation of DIC and IBU but without a clear time-dependent trend; on the other hand, KET concentrations were always below the detection limit. Analyses of a large panel of molecular, biochemical and cellular biomarkers highlighted that all investigated NSAIDs caused alterations of immunological parameters, genotoxic effects, modulation of lipid metabolism and changes in cellular turn-over. This study provided the evidence of long-term ecotoxicological potential of NSAIDs, further unraveling the possible hazard for wild marine organisms.

Transcriptional and cellular effects of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in experimentally exposed mussels, Mytilus galloprovincialis.

The aim of the present investigation was to provide new insights on accumulation and possible adverse effects of various non-steroidal anti-inflammatory drugs (NSAIDs) in mussels, Mytilus galloprovincialis, exposed to an environmentally realistic concentration (0.5µg/L) of individual compounds, Acetaminophen (AMP), Diclofenac (DIC), Ibuprofen (IBU), Ketoprofen (KET) or Nimesulide (NIM). The measurement of drugs in mussel tissues was integrated with both functional alterations at cellular level and transcriptomic responses. Results indicated the capability of mussels to accumulate DIC and NIM, while AMP, IBU and KET were always below detection limit. A large panel of ecotoxicological biomarkers revealed the early onset of alterations induced by tested NSAIDs on immunological responses, lipid metabolism and DNA integrity. The gene transcription analysis through DNA microarrays, supported cellular biomarker results, with clear modulation of a large number of genes involved in the arachidonic acid and lipid metabolism, immune responses, cell cycle and DNA repair. The overall results indicated an ecotoxicological concern for pharmaceuticals in M. galloprovincialis, with transcriptional responses appearing as sensitive exposure biomarkers at low levels of exposure: such changes, however, are not always paralleled by corresponding functional effects, suggesting caution when interpreting observed effects in terms of perturbed cellular pathways. Fascinating similarities can also be proposed in the mode of action of NSAIDs between bivalves and vertebrate species.

Ecotoxicological potential of non-steroidal anti-inflammatory drugs (NSAIDs) in marine organisms: Bioavailability, biomarkers and natural occurrence in Mytilus galloprovincialis.

Pharmaceuticals represent a major environmental concern since the knowledge on their occurrence, distribution and ecotoxicological potential is still limited particularly in coastal areas. In this study, bioaccumulation and cellular effects of various non steroidal anti-inflammatory drugs (NSAIDs) were investigated in mussels Mytilus galloprovincialis to reveal whether common molecules belonging to the same therapeutic class might cause different effects on non target organisms. Organisms exposed to environmental concentrations of acetaminophen (AMP), diclofenac (DIC), ibuprofen (IBU), ketoprofen (KET) and nimesulide (NIM) revealed a significant accumulation of DIC, IBU and NIM, while AMP and KET were always below detection limit. Nonetheless, for all tested NSAIDs, measurement of a large panel of ecotoxicological biomarkers highlighted impairment of immunological parameters, onset of genotoxicity and modulation of lipid metabolism, oxidative and neurotoxic effects. Laboratory results were integrated with a field study which provided the first evidence on the occurrence of DIC, IBU and NIM in tissues of wild mussels sampled during summer months from an unpolluted, touristic area of Central Adriatic Sea. Overall results demonstrated M. galloprovincialis as a good sentinel species for monitoring presence and ecotoxicological hazard of pharmaceuticals in the Mediterranean.

Antioxidant and oxidative stress related responses in the Mediterranean land snail Cantareus apertus exposed to the carbamate pesticide Carbaryl.

The aim of the present work was to study the alterations of the antioxidant defenses and the overall susceptibility to oxidative stress of the terrestrial snail Cantareus apertus exposed to the carbamate pesticide Carbaryl at a low environmentally realistic concentration. The animals were exposed to Lactuca sativa soaked for 1h in 1µM Carbaryl. The temporal dynamics of the responses was assessed by measurements at 3, 7 and 14days of exposure. C. apertus exposed to Carbaryl activates a number of enzymatic antioxidant responses, represented by the early induction of catalase, glutathione peroxidase, glutathione reductase, followed by a delayed induction of superoxide dismutase. Concomitantly, a derangement of the total oxyradical scavenging of the tissues was observed, suggesting an overall impairment of the tissue capability to neutralize ROS probably resulting from the overall negative balance between enzymatic antioxidant defense capability and oxidative stress intensity. This negative balance exposed the animals to the risk of oxidative stress damages including genotoxic damage. Compared to acetylcholinesterase inhibition, the antioxidant responses developed to Carbaryl exposure at the low concentration utilized showed a greater percentage variation in exposed organisms. The results pointed out the high sensitivity of the antioxidant and oxidative stress related responses to Carbaryl exposure at an environmental realistic concentration, demonstrating their usefulness in environmental monitoring and risk assessment. The study highlights also the usefulness of the terrestrial snail C. apertus as potential bioindicator species for assessing the risk of pesticide environmental contamination.

Could molecular effects of Caulerpa racemosa metabolites modulate the impact on fish populations of Diplodus sargus?

The green alga Caulerpa racemosa is a non-native, invasive species in the Mediterranean, and an important stressor for several native organisms. The algal capacity to produce secondary metabolites has been suggested to modulate success of the C. racemosa invasion, although many of potentially involved biological pathways still remain unexplored. In this respect, the aim of the present study was to investigate some molecular and cellular effects in the white seabream Diplodus sargus, an ecologically key species, which included the alien C. racemosa in its diet. Organisms were sampled in 2 seasonal periods from 3 locations of Southern Italy, each characterized by different levels of algal abundance. The level of caulerpin, one of the main secondary algal metabolites, in fish tissues has been used as an indicator of the trophic exposure to the seaweed and related with molecular and cellular responses. Chemical analyses indicated that fish from invaded sites can accumulate caulerpin, with liver concentrations ranging from a few up to hundreds of µg/g. Biomarkers analyses revealed only limited alterations of the main antioxidant defences, such as glutathione reductase and levels of glutathione; on the other hand, increased enzymatic activities of cytochrome P450, glutathione S-transferases and acyl CoA oxidase, as well as enhanced gene transcription for peroxisome proliferator-activated receptor alpha, cytochrome P4501A and vitellogenin 1 were observed in fish more exposed to C. racemosa as indicated by liver concentrations of caulerpin higher than 50 µg/g. Despite a direct molecular relationship with this algal metabolite could not be established, our results suggest that a C. racemosa enriched diet can modulate biotransformation and fatty acids metabolism of D. sargus. Assessing whether similar effects represent short- or long-term effects will be of crucial importance to understand consequences on the general health status and reproductive performance of exposed key fish species in the Mediterranean region.

Effects of harmful dinoflagellate Ostreopsis cf. ovata exposure on immunological, histological and oxidative responses of mussels Mytilus galloprovincialis.

In the last decade massive blooms of the Ostreopsis cf. ovata have occurred in the Mediterranean basin, posing great concern to both environmental and human health. Biotoxicological and chemical studies demonstrated that O. cf. ovata produces palytoxin and ovatoxins; besides direct respiratory effects on humans due to inhalation of marine toxic aerosols, O. cf. ovata blooms can cause adverse effects on benthic invertebrates. The main aim of this study was to highlight the role of immunological, cellular and oxidative mechanisms in modulating the toxicity induced by O. cf. ovata in mussels Mytilus galloprovincialis. Organisms were exposed in laboratory condition to O. cf. ovata and analysed after 7 and 14 days of exposure. Obtained results demonstrated a clear involvement of the immune system with a significant decrement of granulocytes respect to the hyalinocytes type cells, a diminished phagocytosis activity and a reduced lysosomal membrane stability in haemocytes, after both 7 and 14 days of exposure. Histological analyses showed a decrease of the digestive gland wall thickness, dilatation of the tubules, haemocytes infiltration into the digestive gland and a decrement of neutral lipid levels in exposed mussels; similar results suggest a possible inhibition of the feeding activity, with a consequent induction of authophagic phenomena and utilization of stored reserve products such as neutral lipids. Antioxidant parameters revealed a limited role of O. cf. ovata to induce oxidative stress in mussels exposed under laboratory conditions excepting for a certain increase of catalase, glutathione reductase and glutathione peroxidases activities, and a significantly higher capability to neutralize peroxyl radicals in mussels exposed for 14 days. Although the obtained results suggest a non-specific response of mussels to the O. cf. ovata exposure, observed effects on the general health status of exposed mussels should be adequately considered when assessing the ecological relevance of these algal blooms.

Alterations of ROS pathways in scleroderma begin at stem cell level.

Scleroderma is a chronic systemic autoimmune disease (primarily of the skin) characterized by fibrosis (or hardening), vascular alterations and autoantibodies production.There are currently no effective therapies against this devastating and often lethal disorder. Despite the interest for the immunomodulatory effects of mesenchymal stem cells (MSCs) in autoimmune diseases, the role of MSCs in scleroderma is still unknown. A pivotal role in scleroderma onset is played by oxidative stress associated with the accumulation of great amounts of reactive oxygen species (ROS). This study depicts some phenotypic and functional features of MSCs isolated from the skin of healthy and scleroderma patients; the ROS production and accumulation, the expression of ERK1/2 and the effects of the stimulation with PDGF, were analyzed in MSCs; results were compared to those observed in primary fibroblasts (Fbs) isolated from the same subjects. We found that the pro-oxidant environment exerted by scleroderma affects MSCs, which are still able to counteract the ROS accumulation by improving the antioxidant defenses. On the contrary, scleroderma fibroblasts show a disruption of these mechanisms, with consequent ROS increase and the activation of the cascade triggered by scleroderma auto-antibodies against PDGFR.

Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: a multibiomarkers approach with mussels Mytilus galloprovincialis.

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans. The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na(+)/K(+)-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage). Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na(+)/K(+)-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.

Effect of biologic therapies targeting tumour necrosis factor-α on cutaneous mesenchymal stem cells in psoriasis.

BACKGROUND: Psoriasis is a Th1 immune-mediated, inflammatory disease, in which skin lesions appear many years before the related metabolic and cardiovascular comorbidities, according to the theory of the 'psoriatic march'. Inducible nitric oxide synthetase (iNOS), tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) are directly implicated in determining both skin lesions and systemic involvement in psoriasis. Reactive oxygen species actively promote the secretion of inflammatory Th1 cytokines directly involved in the pathogenesis of psoriasis. OBJECTIVES: Evaluation of VEGF expression and production, nitric oxide (NO) production, iNOS expression, and the antioxidant response of mesenchymal stem cells (MSCs), both before and after 12 weeks of treatment with the TNF-α inhibitors adalimumab or etanercept. METHODS: Biochemical, morphological and immunohistochemical analyses were performed in MSCs isolated from nonlesional, perilesional and lesional skin of patients with psoriasis, before and after treatment. RESULTS: The treatments were able to reduce the expression and production of VEGF, the expression of iNOS and the production of NO in MSCs of patients with psoriasis. TNF-α inhibitors also reduced the oxidative damage in MSC membrane and proteins, several antioxidant systems responded to treatments with a general inhibition of activities (glutathione S-transferase and catalase) and these effects were also supported by a general decrease of total oxyradical scavenging capacity towards hydroxyl radicals and peroxynitrite. CONCLUSIONS: TNF-α inhibitors are able to change the physiopathological pathway of psoriasis, and our results suggest their therapeutic effects already take place at the level of MSCs, which probably represent the cells primarily involved in the 'psoriatic march'.

Oxidative stress defense in human-skin-derived mesenchymal stem cells versus human keratinocytes: Different mechanisms of protection and cell selection.

Stem cells are undifferentiated cells with the capacity for self-renewal and differentiation. Here we have determined the susceptibility to oxidative stress of isolated mesenchymal stem cells from human skin (S-MSCs) in comparison with keratinocytes, which are differentiated cells of the same lineage. To induce pro-oxidant conditions, S-MSCs and keratinocytes were exposed to 0.5mM H(2)O(2) for 2 h, with oxidative effects analyzed after 4, 12, 24, and 48 h of recovery, in terms of cell growth, vitality, apoptosis, DNA damage, variations in individual antioxidant defense and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals. The data indicate different abilities across these two cell types to counteract this oxidative stress, which reflects stress that would normally be experienced by these cells under basal conditions. Human keratinocytes seem to have much greater antioxidant defense to counteract the oxidative injury to which they are continuously exposed in the skin. The S-MSCs are surrounded by a complex microenvironment that protects them from external insults, and so they do not have a particularly efficient defense system, and they were generally less responsive to enhanced pro-oxidant challenge. S-MSCs seem particularly prone to apoptotic events, which might thus represent their primary defense mechanism against stress.

Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach.

An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.

Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla.

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon.

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.