Impact of aged and virgin polyethylene microplastics on multi end-points effects of freshwater fish tissues.

The buildup of plastic waste in aquatic environments presents serious threats to the environment, wildlife, and ultimately to humans. Specifically, microplastics (MPs) ingestion by aquatic animals leads to adverse physiological and toxicological effects. In addition, discarded MPs undergo aging and degradation processes which affect their morphological properties and chemical composition, enhancing the absorption of environmental pollutants. Under this prism, the present research was conducted to investigate and compare the impact of 'aged' versus pristine low-density polyethylene microplastics (PE-MPs) on various toxicity endpoints as biochemical and molecular parameters in the muscle tissue and liver of the freshwater fish species Perca fluviatilis. In parallel, the morphological, physicochemical, and structural changes occurred in "aged" PE-MPs, (after being exposed to UV radiation for 120 days) were studied, significantly illustrating signs of oxidation and crack propagation at the surface of the studied MPs. Fish were exposed to artificial diet reached with virgin and "aged" PE-MPs, sized 100-180 µm, at concentrations of 1 mg/g of dry food for a period of 15-days. Thereafter, liver and muscle tissues were analyzed in relation to multi oxidative parameters. Compared to the control group, the observed changes in the examined fish included increased activities of antioxidant enzymes, as superoxide dismutase, catalase and glutathione reductase, enhanced concentrations of malondialdehyde, protein carbonylation, HSP70 levels, elevated MAPK phosphorylation, induction of ubiquitin-proteins, as well as heightened levels of Bax/Bcl-2 proteins, caspases and differentiated levels of LC3 II/I, SQSTM1/p62. From the studied biomarkers, apoptosis, ubiquitin and hsp70 levels, showed a more sensitive response against the ingested MPs, followed by autophagy, p38MAPK levels, antioxidant enzymes, MDA and carbonylation levels. The effect of "aged" PE-MPs was more pronounced compared to that of the virgin ones. When evaluating the response of all oxidative stress biomarkers across the studied tissues, the liver demonstrates the highest response for the majority of the biomarkers against both virgin and "aged" PE-MPs. These findings strongly indicate that "aged" MPs activate the antioxidant defence mechanisms and impact the cellular well-being of the examined fish species.

Dose-Dependent Cytotoxicity of Polypropylene Microplastics (PP-MPs) in Two Freshwater Fishes.

The massive accumulation of plastics over the decades in the aquatic environment has led to the dispersion of plastic components in aquatic ecosystems, invading the food webs. Plastics fragmented into microplastics can be bioaccumulated by fishes via different exposure routes, causing several adverse effects. In the present study, the dose-dependent cytotoxicity of 8−10 µm polypropylene microplastics (PP-MPs), at concentrations of 1 mg/g (low dose) and 10 mg/g dry food (high dose), was evaluated in the liver and gill tissues of two fish species, the zebrafish (Danio rerio) and the freshwater perch (Perca fluviatilis). According to our results, the inclusion of PP-MPs in the feed of D. rerio and P. fluviatilis hampered the cellular function of the gills and hepatic cells by lipid peroxidation, DNA damage, protein ubiquitination, apoptosis, autophagy, and changes in metabolite concentration, providing evidence that the toxicity of PP-MPs is dose dependent. With regard to the individual assays tested in the present study, the biggest impact was observed in DNA damage, which exhibited a maximum increase of 18.34-fold in the liver of D. rerio. The sensitivity of the two fish species studied differed, while no clear tissue specificity in both fish species was observed. The metabolome of both tissues was altered in both treatments, while tryptophan and nicotinic acid exhibited the greatest decrease among all metabolites in all treatments in comparison to the control. The battery of biomarkers used in the present study as well as metabolomic changes could be suggested as early-warning signals for the assessment of the aquatic environment quality against MPs. In addition, our results contribute to the elucidation of the mechanism induced by nanomaterials on tissues of aquatic organisms, since comprehending the magnitude of their impact on aquatic ecosystems is of great importance.

The cytotoxicity effect of a bis-MPA-based dendron, a bis-MPA-PEG dendrimer and a magnetite nanoparticle on stimulated and non-stimulated human blood lymphocytes.

Dendrimers and dendrons offer a high surface area and nanoscale size and magnetic nanoparticles can be easily detected and manipulated due to their magnetic properties. The aim of the present study is to investigate the in vitro toxicity of Polyester-8-hydroxyl-1-carboxyl bis-MPA dendron, generation 3 (bis-MPA), Hyperbranched G4-PEG6k-OH (PEG) dendrimer and magnetite nanoparticle (Fe3O4), in human lymphocytes. Cell viability assays were performed on non-stimulated and lipopolysaccharide (LPS) stimulated lymphocytes, after exposure to various concentrations of the nanoparticles, using the Trypan blue assay, Flow Cytometry with 7-Amino Actinomycin D fluorescent dye (7-AAD), as well as the 3-[4,5-dimethylthiazol-2-yl] 2,5 diphenyl tetrazolium bromide (MTT) colorimetric method. The results collectively showed that after 24 h both the dendron and dendrimer at 50 µM concentration exhibited low cytotoxicity to non-stimulated and stimulated lymphocytes. Magnetite nanoparticle (Fe3O4) in concentrations 50-1000 µg/mL revealed negligible cytotoxicity to stimulated and non-stimulated lymphocytes. Moreover, the amount of intercellular Reactive Oxygen Species with or without treatment was assessed by means of the DCFH-DA to evaluate the presence of any oxidative stress. We propose herein simple cytotoxicity tests which indicate that these nanoparticles, after further studying, can serve as ideal drug carriers.

Do poly(lactic acid) microplastics instigate a threat? A perception for their dynamic towards environmental pollution and toxicity.

Fears concerning microplastics (MPs) environmental fate and persistence are progressively expanding on a global basis, with the emphasis given to manufacturing bioplastics for substituting petro-derived plastics extensively growing. Among them, poly(lactic acid) (PLA) holds a pioneering role towards the replacement of conventional polymeric materials, owing to its multifunctional properties, enclosing superior mechanical properties, low cost, renewability, great biocompatibility, transparency, and thermoplasticity launching many fields of application. Due to the wide applicability of PLA in several sectors of everyday life, its waste to be released into the environment is expected to follow a growing tendency during the upcoming years. Even though PLA is a biodegradable polyester, it actually degrades under specific composting environments, including a rich oxygen environment with high temperatures (58-80 °C), high humidity (>60% moisture) as well as the presence of micro-organisms (thermophilic bacteria). Additionally, in various studies it has been implied that PLA displays slower degradation performance when found in blends with other conventional polymers, underlining the unspecified effects on PLA degradation profile, keeping thus the information about PLA degradation from a blur standpoint. Therefore, a deepened understanding of the fate and dynamic effects of PLA MPs is of primary importance. Nevertheless, the current examination of the effects of PLA MPs in terms of sorption capacities and toxicity is so far limited and broadly unexplored since the current scientific emphasis has been merely centered on the conventional MPs' behavior. In this light, the present review provides an inclusive overview of the ongoing research of poly(lactic acid) in the framework of microplastics' pollution, while the future trends and missing points in this context are highlighted.

Differentiation in the expression of toxic effects of polyethylene-microplastics on two freshwater fish species: Size matters.

The built up of microplastic (MPs) remains is shaping a new aquatic habitat and imposes the necessity for research of the effects that these relatively new pollutants exert on organisms, environment, and human health. The purpose of the present study was to verify if there is a particle-size dependence of fish response to MPs. Thus, we exposed two freshwater fish species, the zebrafish (Danio rerio) and perch (Perca fluviatilis) for 21 days to polyethylene microplastics (PE-MPs) sized 10-45 µm and 106-125 µm. Thereafter, in the liver and gills tissues, biochemical and molecular parameters and the metabolic profile were examined. Ex-vivo characterization by ATR-FTIR spectroscopy exhibited increased concentration of 10-45 µm PE-MPs in the liver of the two fish species while 106-125 µm PE-MPs mostly concentrated in fish gills. The penetration of PE-MPs to fish and the induced oxidative stress triggered changes in lipid peroxidation, DNA damage and ubiquitination and furthermore stimulated signal transduction pathways leading to autophagy and apoptosis. The smaller PE-MPs were more potent in inducing alterations to all the latter parameters measured than the larger ones. Tissue response in both fish seems to depend on the parameter measured and does not seem to follow a specific pattern. Our results showed that there is no clear sensitivity of one fish species versus the other, against both sizes of PE-MPs they were exposed. In perch the metabolic changes in gills were distinct to the ones observed in liver, following a size dependent pattern, indicating that stress conditions are generated through different mechanisms. All the parameters employed can be suggested further as biomarkers in biomonitoring studies against PE-MPs.

Toxicity and Functional Tissue Responses of Two Freshwater Fish after Exposure to Polystyrene Microplastics.

Microplastics (MPs)' ingestion has been demonstrated in several aquatic organisms. This process may facilitate the hydrophobic waterborne pollutants or chemical additives transfer to biota. In the present study the suitability of a battery of biomarkers on oxidative stress, physiology, tissue function and metabolic profile was investigated for the early detection of adverse effects of 21-day exposure to polystyrene microplastics (PS-MPs, sized 5-12 µm) in the liver and gills of zebrafish Danio rerio and perch, Perca fluviatilis, both of which are freshwater fish species. An optical volume map representation of the zebrafish gill by Raman spectroscopy depicted 5 µm diameter PS-MP dispersed in the gill tissue. Concentrations of PS-MPs close to the EC50 of each fish affected fish physiology in all tissues studied. Increased levels of biomarkers of oxidative damage in exposed fish in relation to controls were observed, as well as activation of apoptosis and autophagy processes. Malondialdehyde (MDA), protein carbonyls and DNA damage responses differed with regard to the sensitivity of each tissue of each fish. In the toxicity cascade gills seemed to be more liable to respond to PS-MPs than liver for the majority of the parameters measured. DNA damage was the most susceptible biomarker exhibiting greater response in the liver of both species. The interaction between MPs and cellular components provoked metabolic alterations in the tissues studied, affecting mainly amino acids, nitrogen and energy metabolism. Toxicity was species and tissue specific, with specific biomarkers responding differently in gills and in liver. The fish species that seemed to be more susceptible to MPs at the conditions studied, was P. fluviatilis compared to D. rerio. The current findings add to a holistic approach for the identification of small sized PS-MPs' biological effects in fish, thus aiming to provide evidence regarding PS-MPs' environmental impact on wild fish populations and food safety and adequacy.

Insights into the toxicity of biomaterials microparticles with a combination of cellular and oxidative biomarkers.

Considering that the extensive biomedical, pharmaceutics, cosmetic and other industrial applications of biomaterials (BMs) is of great concern nowadays, regarding their environmental risk, the present study aimed to investigate the effects of four BMs, poly(ε-caprolactone) (PCL), poly(butylene succinate) (PBSu), chitosan (CS) and modified chitosan (succinic acid grafted chitosan) (CS-Suc) in the form of microplastics (particle sizes less than 1 mm) on biochemical parameters of snails Cornu aspersum hemocytes. Due to the absence of knowledge about the environmentally relevant concentrations of BMs, snails were initially treated through their food with a wide range of nominal concentrations of each BM to define the half maximal effective concentration (NRRT50), according to the destabilization degree of hemocytes' lysosomal membranes (by mean of neutral red retention time/NRRT assay). Thereafter, snails were treated with each BM, at concentrations lower than the estimated NRRT50 values in all cases, for periods up to 15 days. After the end of the exposure period, a battery of stress indices were measured in hemocytes of challenged snails. According to the results, all parameters tested in BMs-treated snails statistically differed from those measured in BMs-free snails, thus indicating the pro-oxidant potential of BMs, as well as their ability to affect animals' physiology. The most considerable effect in most cases seems to be caused by modified chitosan and PCL, while chitosan appears to be the least toxic. A common response mechanism of snails' blood cells against the 4 BMs used in the present study was shown. After exposure to each of the studied BMs a significant augmentation in protein carbonyls, MDA equivalents and DNA damage, while a significant reduction in NRRT values was determined in the snails hemocytes, in relation to the unexposed animals. From the biochemical parameters examined, MDA equivalents and DNA damage seem to be more susceptible than the other parameters studied, to respond to BMs effect, with MDA to react with more sensitivity to PCL and CS, while DNA damage to CS-Suc and PBSu. Our results could suggest the simultaneous use of the latter biomarkers in biomonitoring studies of terrestrial ecosystems against the specific BMs.

Toxicity assessment and comparison of the land snail's Cornu aspersum responses against CuO nanoparticles and ZnO nanoparticles.

The goal of the present study was to examine the effects of ZnO NPs and CuO NPs on Cornu aspersum land snail, enlightening their cytotoxic profile. ZnO NPs and CuO NPs were synthesized and thoroughly characterized. Α series of concentrations of either ZnO NPs or CuO NPs were administered in the feed of snails for 20 days. Thereafter, neutral red retention assay was conducted, in order to estimate NRRT50 values. Subsequently, snails were fed with NPs concentrations slightly lower than the concentrations that were corresponding to the NRRT50 values, i.e. 3 mg·L-1 ZnO NPs and 6 mg·L-1 CuO NPs, for 1, 5, 10 and 20 days. Both NPs agglomerates were detected in hemocytes by Transmission Electron Microscopy. Moreover, both effectors resulted to toxicity in the snails' hemocytes. The latter was shown by changes in the NRRT50 values, increased reactive oxygen species (ROS) production, lipid peroxidation, DNA integrity loss, protein carbonyl content, ubiquitin conjugates and cleaved caspases conjugates levels compared to the untreated animals. Although ZnO NPs exhibited higher toxicity, as indicated by the NRRT50 values, both NPs affected similarly a wide range of the cellular parameters mentioned above. The latter parameters could constitute sensitive biomarkers in biomonitoring studies of terrestrial environment against nanoparticles.

Common mechanisms activated in the tissues of aquatic and terrestrial animal models after TiO2 nanoparticles exposure.

Titanium dioxide nanoparticles (TiO2-NPs) are among the most popular manufactured and widely used nanoparticles. They are released into the environment, affecting terrestrial and aquatic ecosystems, with unexpected consequences to organisms and human health. The present study investigates the mediated toxicity imposed to the freshwater fish species, zebrafish (Danio rerio) and the prussian carp (Carassius gibelio), and to the terrestrial land snail Cornu aspersum, after their exposure to sublethal concentrations of TiO2-NPs. Oxidative, proteolytic, genotoxic and apoptotic parameters in fish liver and gills, as well as on snail hemocytes were studied and the swimming performance was estimated in order to (a) estimate and suggest the most susceptible animal, and (b) propose a common battery of biomarkers as the most suitable indicator for biomonitoring studies against TiO2-NPs. Our in vivo experiments demonstrated that NPs induced detrimental effects on animal physiology and swimming behavior, while no general pattern was observed in species and tissues responsiveness. Generally, TiO2-NPs seemed to activate a group of molecules that are common for aquatic as well as terrestrial animals, implying the existence of a conserved mechanism. It seems that after exposure to TiO2-NPs, a common mechanism is activated that involves the stimulation of immune system with the production of ROS, damage of lysosomal membrane, protein carbonylation, lipid peroxidation, DNA damage, following proteolysis by ubiquitin and finally apoptosis. Thus, the simultaneous use of the latter biomarkers could be suggested as a reliable multi parameter approach for biomonitoring of aquatic and terrestrial ecosystems against TiO2-NPs.

Magnetite nanoparticles effects on adverse responses of aquatic and terrestrial animal models.

Among pollutants, nanoparticles (NPs) consist a potential environmental hazard, as they could possibly harm the aquatic and terrestrial ecosystems while having unpredictable repercussions on human health. Since monitoring the impact of NPs on aquatic and terrestrial life is challenging, due to the differential sensitivities of organisms to a given nanomaterial, the present study examines magnetite nanoparticles' mediated toxicity in different animal models, representing distinctive environments (terrestrial and aquatic). Oxidative, proteolytic and genotoxic effects were evaluated on the hemocytes of the snail Cornu aspersum; in addition to those, apoptotic effects were measured in gills and liver of the zebrafish Danio rerio, and the prussian carp Carassius gibelio. All biochemical parameters studied increased significantly in animals after 8 days exposure to NPs. Inter-species and inter-tissues differences in responses were evident. Our results suggest a common toxicity response mechanism functioning in the tissues of the three animals studied that is triggered by magnetite NPs. The simultaneous use of these parameters could be established after further investigation as a reliable multi-parameter approach for biomonitoring of terrestrial and aquatic ecosystems against magnetite nanoparticles. Additionally, the results of our study could contribute to the design of studies for the production and rational utilization of nanoparticles.

Differentiation Capacity of Monocyte-Derived Multipotential Cells on Nanocomposite Poly(e-caprolactone)-Based Thin Films.

Background: Μonocyte-derived multipotential cells (MOMCs) include progenitors capable of differentiation into multiple cell lineages and thus represent an ideal autologous transplantable cell source for regenerative medicine. In this study, we cultured MOMCs, generated from mononuclear cells of peripheral blood, on the surface of nanocomposite thin films. Methods: For this purpose, nanocomposite Poly(e-caprolactone) (PCL)-based thin films containing either 2.5 wt% silica nanotubes (SiO2ntbs) or strontium hydroxyapatite nanorods (SrHAnrds), were prepared using the spin-coating method. The induced differentiation capacity of MOMCs, towards bone and endothelium, was estimated using flow cytometry, real-time polymerase chain reaction, scanning electron microscopy and fluorescence microscopy after cells' genetic modification using the Sleeping Beauty Transposon System aiming their observation onto the scaffolds. Moreover, Wharton's Jelly Mesenchymal Stromal Cells were cultivated as a control cell line, while Human Umbilical Vein Endothelial Cells were used to strengthen and accelerate the differentiation procedure in semi-permeable culture systems. Finally, the cytotoxicity of the studied materials was checked with MTT assay. Results: The highest differentiation capacity of MOMCs was observed on PCL/SiO2ntbs 2.5 wt% nanocomposite film, as they progressively lost their native markers and gained endothelial lineage, in both protein and transcriptional level. In addition, the presence of SrHAnrds in the PCL matrix triggered processes related to osteoblast bone formation. Conclusion: To conclude, the differentiation of MOMCs was selectively guided by incorporating SiO2ntbs or SrHAnrds into a polymeric matrix, for the first time.

Biochemical and molecular responses of cyprinids in two Mediterranean lacustrine ecosystems: Opportunities for ecological assessment and biomonitoring.

Lacustrine ecosystems have been altered by accelerating pollution, excessive nutrient and organic load, water abstraction, and are susceptible to climate change. Hence, suggesting sensitive and reliable biomarkers for early assessments of their status is of urgent need. In this study, two freshwater commercial fish species, Cyprinus carpio (carp) and Carassius gibelio (prussian carp) from two lakes (i.e. Koronia and Volvi, Northern Greece) with different anthropogenic pressures were used and a battery of biochemical and molecular biomarkers related to stress response were analyzed in fish gills and liver. In parallel, water physicochemical parameters (T, DO, pH, conductivity, salinity), BOD5 and nutrient (N-NO3, N-NO2, N-NH4, P-PO4) concentrations were measured. Results showed that Lake Koronia had higher conductivity and salinity values and N-NO2 concentrations. Levels of Heat Shock Response (HSR), MAPK phosphorylation, protein carbonylation, lipid peroxidation products, Bax/Bcl-2 ratio, ubiquitination and caspases were increased in gills and liver of both fish species sampled from Lake Koronia in relation to those of Lake Volvi. Likewise, liver lipid content was increased in both fish species sampled from Lake Koronia compared to those sampled from Lake Volvi. The results indicate and reflect the higher environmental degradation that prevails in Lake Koronia ecosystem in comparison to that of Lake Volvi. The fish species studied showed different susceptibility depending on the biomarkers examined. In addition, our results from both examined species provide insight into the mechanisms involved in acclimatization to stressful environments and support the role of the studied biomarkers as sensitive and reliable tools for ecological assessments of lake ecosystems in biomonitoring studies.

Insights into the toxicity of iron oxides nanoparticles in land snails.

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L-1 for IONPs and 1 mg L-1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future.

Human mesenchymal stem cells with enhanced telomerase activity acquire resistance against oxidative stress-induced genomic damage.

BACKGROUND: Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative senescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. METHODS: Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). RESULTS: We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superoxide dismutase and catalase, than control MSCs. DISCUSSION: On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell's basic antioxidant machinery.

Toxicity assessment and comparison between two types of iron oxide nanoparticles in Mytilus galloprovincialis.

Nanoparticles (NPs), due to their increased application and production, are being released into the environment with unpredictable impact on the physiology of marine organisms, as well as on entire ecosystems and upcoming effects on human health. The aim of the present study was to evaluate and compare the oxidative responses of the mussel Mytilus galloprovincialis after exposure to iron oxide NPs and to iron oxide NPs incorporated into zeolite for 1, 3 and 7 days. Our results showed that both effectors induced changes on animal physiology by causing oxidative stress in hemocytes of exposed mussels compared to control animals. This was shown by the significant increase in reactive oxygen species (ROS) production, protein carbonylation, lipid peroxidation, ubiquitin conjugates and DNA damage. In addition an increase in prooxidant levels as measured by the prooxidant-antioxidant balance (PAB) assay was observed in exposed mussels' hemolymph. The results show that ROS, DNA damage, protein and lipid oxidation, ubiquitin conjugates and PAB could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors. In addition, more studies are needed in order to ensure the safety of these NPs on various biomedical applications, since it is critical to design NPs that they meet the demands of application without causing cellular toxicity.

Effects of cadmium and 17ß-estradiol on Mytilus galloprovincialis redox status. Prooxidant-antioxidant balance (PAB) as a novel approach in biomonitoring of marine environments.

Cadmium and 17ß-estradiol are rapidly accumulated in mussel tissues, making mussels excellent pollution sentinel organisms. The aim of the present study was to compare the oxidative responses of the mussels after 1, 3 and 7 days of exposure to cadmium with those to 17ß-estradiol and subsequently, to suggest a multi-parametric approach for biomonitoring studies. Our results showed that environmentally relevant concentrations of either cadmium or 17ß-estradiol for 1, 3 and 7 days induced oxidative stress in hemocytes of exposed mussels. The latter was determined by significantly increased ROS levels and apoptosis, by suppression of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) expression levels and subsequent increased prooxidant levels, as measured by prooxidant-antioxidant balance (PAB) assay. To our knowledge this is the first time that prooxidant-antioxidant balance is evaluated in invertebrates as an index of oxidative stress. The simultaneous use of the parameters of prooxidant-antioxidant balance and antioxidant enzymes expression patterns, in combination with ROS production levels and apoptosis, in mussel hemocytes is suggested as an approach that may help to better evaluate the impact of environmental pollution on marine organisms and thereupon ecosystems.

Oxidative stress parameters induced by exposure to either cadmium or 17ß-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules.

The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17ß-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring. Our results suggest that micromolar concentrations of either cadmium or 17ß-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5 µM of cadmium chloride or with 25 nM of 17ß-estradiol for 30 min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17ß-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3K) and reaching Na(+)/H(+) exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca(2+)-dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and cyclic adenosine-3'-5'-monophosphate (cAMP). Our results also attribute a protective role to cAMP, since pre-elevated intracellular cAMP levels inhibited the signal induced by each exposure. Finally, since aquatic invertebrates have been the most widely used monitoring organisms for pollution impact evaluation in marine environments and taking under consideration the positive correlation obtained between the studied parameters, we can suggest the simultaneous use of these oxidative stress parameters offering an effective early warning system in biomonitoring of aquatic environments.

Antioxidant and pro-oxidant challenge of tannic acid in mussel hemocytes exposed to cadmium.

The present study investigates the antioxidant and pro-oxidant behavior of tannic acid (TA) in hemocytes of mussel Mytilus galloprovincialis, in the presence or the absence of cadmium (Cd). TA at concentrations up to 20 µM, primarily found to be no toxic (in terms of cell viability, superoxide anions, nitric oxide and lipid peroxidation products currently estimated), significantly diminished the cytotoxic and oxidative effects induced by the metal (50 and/or 100 µM) in all cases. On the other hand, higher concentrations of TA (40 and 60 µM) were toxic, thus enhancing Cd-mediated cytotoxic and oxidative effects. The present study showed TA beneficiary properties in hemocytes of mussels, at least at low concentrations, while TA at concentrations higher than 20 µM could serve as an excellent oxidized substrate, thus enhancing toxic effects either alone or with the presence of micromolar concentrations of non transition metals, such as Cd.

Effect of 17ß-estradiol on adhesion of Mytilus galloprovincialis hemocytes to selected substrates. Role of alpha2 integrin subunit.

The process of hemocyte adhesion to extracellular matrix (ECM) proteins plays a crucial role in cell immunity. In most of these interactions between ECM proteins and cells, integrins are involved. The results of the present study showed that incubation of Mytilus galloprovincialis hemocytes with 17ß-estradiol caused significant increased adhesion of hemocytes to ECM proteins and specifically to laminin-1, collagen IV and oxidized collagen IV, in relation to control cells. The adhesion of hemocytes to oxidized collagen was significantly higher than to either collagen IV or to laminin-1. In accordance with this, inhibition of either NADPH oxidase or nitric oxide (NO) synthase attenuated 17ß-estradiol effect on hemocyte adhesion, suggesting that the high levels of free radicals, produced after 17ß-estradiol effect, could contribute to the high adhesion of hemocytes to laminin-1 and collagen IV. The implication of ROS was further confirmed by the use of the oxidant rotenone, which caused elevation of cell adhesion in relation to control and by the antioxidant NAC which attenuated 17ß-estradiol effect. The mechanism of 17ß-estradiol induced adhesion to laminin-1, collagen IV and oxidized collagen IV involves a large number of intracellular components, as Na+/H+ exchanger (NHE), all isoforms of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K) and c-jun N-terminal kinase (JNK) as well as alpha2 integrin subunit. Maintenance of high cyclic adenosine-3'-5'-monophosphate (cAMP) levels caused non significant higher adhesion of hemocytes to ECM proteins in relation to control cells. Our results showed that 17ß-estradiol caused a significant increase in α2 integrin subunit levels, which was reduced after inhibition of NHE, PI3K, PKC, NO synthase, NADPH oxidase and JNK. In addition, our results showed that apart from 17ß-estradiol, high cAMP and high ROS levels caused significantly higher induction of α2 integrin subunit levels in relation to control. Our results imply a potential involvement of cAMP in immune responses of Mytilus hemocytes, which needs further investigation.

NHE-1 and ß1 integrin dependent monocyte adhesion and migration after glucose, insulin or PPARγ stimulation.

In the present study the effect of high glucose concentrations, insulin, PPARγ activators (rosiglitazone) and NHE-1 inhibitors (cariporide) in atherosclerosis-related functions of human monocytes was investigated. Monocyte adhesion to laminin-1, collagen type IV and endothelial cells, as well as monocyte migration through the same substrates were studied. Incubation of the monocyte suspension with high glucose concentrations, insulin and rosiglitazone induced all the studied atherosclerosis-related functions of the monocytes. In all these functions the addition of cariporide counteracted the activity of glucose, insulin and rosiglitazone. The use of antigen for ß1 integrin also counteracted the activity of the above in monocyte adhesion in all three substrates. The data of the present study suggests that PPARγ activation in monocytes induces atherosclerosis, and that NHE-1 and ß1 integrin play an important role in the beginning of atherosclerosis.