•CdS QDs were cytotoxic for mussel hemocytes and gill cells in vitro.•Ionic Cd was the most toxic form, followed by CdS QDs and bulk CdS.•CdS QDs altered oxidative balance and caused DNA damage in ...mussel cells.•CdS QDs caused a particle-specific immunostimulation on phagocytosis of hemocytes.•Conceptual models for cellular handling and toxicity of CdS QDs are proposed.
CdS quantum dots (QDs) show a great promise for treatment and diagnosis of cancer and for targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. In spite of their advantages it is important to determine if CdS QDs can exert toxicity on biological systems. In the present work, cytotoxicity of CdS QDs (5nm) at a wide range of concentrations (0.001–100mg Cd/L) was screened using neutral red (NR) and thiazolyl blue tetrazolium bromide (MTT) assays in isolated hemocytes and gill cells of mussels (Mytilus galloprovincialis). The mechanisms of action of CdS QDs were assessed at sublethal concentrations (0.31–5mg Cd/L) in the same cell types through a series of functional in vitro assays: production of reactive oxygen species (ROS), catalase (CAT) activity, DNA damage, lysosomal acid phosphatase (AcP) activity, multixenobiotic resistance (MXR) transport activity, Na-K-ATPase activity (only in gill cells) and phagocytic activity and damage to actin cytoskeleton (only in hemocytes). Exposures to CdS QDs lasted for 24h and were performed in parallel with exposures to bulk CdS and ionic Cd. Ionic Cd was the most toxic form to both cell types, followed by CdS QDs and bulk CdS. ROS production, DNA damage, AcP activity and MXR transport were significantly increased in both cell types exposed to the 3 forms of Cd. CAT activity increased in hemocytes exposed to the three forms of Cd while in gill cells only in those exposed to ionic Cd. No effects were found on hemocytes cytoskeleton integrity. Effects on phagocytosis were found in hemocytes exposed to bulk CdS and to CdS QDs at concentrations equal or higher than 1.25mg Cd/L but not in those exposed to ionic Cd, indicating a particle-specific effect on phagocytosis. In conclusion, cell-mediated immunity and gill cell function represent significant targets for CdS QDs toxicity.
Within the frame of the 2nd Iberian Congress of Environmental Contamination and Toxicology (University of the Basque Country, Leioa, June 1998) a workshop was held about the use of biomarkers in ...marine pollution monitoring. Among others, the following biomarkers received special attention: metallothionein induction, acetylcholinesterase inhibition, cytochrome P450 system induction, imposex, lysosomal enlargement and lysosomal membrane destabilisation, and peroxisome proliferation. These biomarkers can be used to evaluate exposure to and effect of different contaminants (metals, organic xenobiotics and organometallic compounds) and they can be measured using different methodological approaches (biochemistry, cytochemistry, immunochemical methods based on the use of biotechnology). Before the application of a set of biomarkers in pollution monitoring programmes, well-defined protocols of Quality Assurance have to be established to allow adequate comparison of results. It is also necessary to include analysis of standard reference materials and to obtain detailed knowledge of basal values and seasonal variations of the biomarkers in various species, as well as to integrate the information obtained with the different biomarkers. Marine bivalve molluscs such as mussels are appropriate sentinel species for most of the biomarkers proposed except for the induction of the cytochrome P450 system, which should be measured in fish, and the degree of imposex, which is a biomarker of exposure to TBT specifically measured in certain gastropod molluscs. As a result of the workshop, a battery of biomarkers of contaminant exposure and effects are proposed that could be incorporated into programmes monitoring the quality of the coastal environment in the Iberian Peninsula. These measures would be undertaken in conjunction with chemical measures of contaminant burdens in selected sentinel species.
Endocrine disrupting chemicals (EDCs) interfere with the functioning of the endocrine system, causing reproductive and developmental disturbances in aquatic wildlife. Appearance of intersex gonads ...and elevated plasma levels of vitellogenin in male fish are well known biomarkers of exposure to xenoestrogenic EDCs. In the present study, intersex condition and transcription levels of vtg and cyp19a1b were assessed in five thicklip grey mullet populations from the Basque coast (Bay of Biscay). Levels of EDCs (estrogenic hormones, polycyclic musks, bisphenol-A, phthalates, alkylphenols and pesticides) were determined in water and fish bile. Intersex gonads were observed in three out of five mullet populations. Vtg and cyp19a1b were up-regulated in mullet populations with relatively higher EDCs load. Phthalates and pesticides were the most abundant EDCs in bile, followed by alkylphenols, musks, bisphenol-A and estrogenic hormones. Statistically significant correlations were found between concentrations of individual and total EDCs in bile and water samples and transcription levels of vtg and cyp19a1b.
•We studied 5 mullet (Chelon labrosus) populations from the Basque coast (Bay of Biscay).•Endocrine disrupting compounds were determined in water and bile samples.•Bile contained phthalates, pesticides, alkylphenols, bisphenol A, estrogenic hormones and musks.•Intersex condition was detected in 3 out of 5 populations.•Up-regulation of vtg and cyp19a1b was observed in male and intersex fish.
In the last years, applications for silver nanoparticles (Ag NPs) continue to increase together with the concerns about their potential input and hazards in aquatic ecosystems, where microalgae are ...key organisms. The aim of the present study was to assess the relative sensitivity of three marine microalgae species with differences in cell wall composition/structure exposed to Poly N-vinyl-2-pirrolidone/Polyethyleneimine (PVP/PEI) coated 5nm Ag NPs and uncoated 47nm Ag NP. As limited attention has been paid to the role of coating agents in NP toxicity, the effect of PVP/PEI alone was also evaluated. After 72h in artificial seawater, 47nm Ag NPs formed around 1400nm size aggregates while PVP/PEI coated 5nm Ag NPs reached around 90nm. Ag+ release in seawater was around 3% for 47nm Ag NPs and 30% for PVP/PEI coated 5nm Ag NPs. PVP/PEI coated 5nm Ag NP aggregates entrapped the algal cells in a network of heteroaggregates, while uncoated 47nm Ag NPs interacted to a lesser extent with algae. The concentration of PVP/PEI coated 5nm Ag NPs that exerted the median effect (EC50) on algae growth pointed out differences in algae sensitivity: T. suecica was about 10 times more sensitive than I. galbana and P. tricornutum. Further, the coating agent alone was as toxic to algae as PVP/PEI coated 5nm Ag NPs, suggesting that presence of the coating agent was the main driver of toxicity of coated NPs. Uncoated 47nm Ag NPs instead, showed similar toxicity towards algae although P. tricornutum was slightly less sensitive than T. suecica and I. galbana, which agrees with the presence of a resistant silicified cell wall in the diatom. The present work demonstrates differences in sensitivity of three marine microalgae, possibly related to their cell surface and size characteristics.
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•Effects of coated, uncoated Ag NPs and coating agent on microalgae were evaluated.•Coated 5 nm Ag NPs produced aggregates of smaller size and released 10 times more Ag ions than uncoated 47 nm Ag NPs.•Coated 5 nm Ag NPs were more toxic than uncoated 47 nm Ag NPs.•The coating agent alone was as toxic as coated 5 nm Ag NPs.•Tetraselmis suecica was about 10 times more sensitive than Isochrysis galbana and Phaeodactylum tricornutum.
Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and ...perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor α (pparα) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARα mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor α was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action.
The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO
2
nanoparticles (NPs) ...in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO
2
NPs (60 and 180 nm) or bulk TiO
2
, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO
2
NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO
2
NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO
2
NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO
2
NPs although more markedly after exposure to TiO
2
-60 NPs. Exposure to bulk TiO
2
and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO
2
and DSLS.
•Effects of treated and untreated urban discharges were assessed in caged mussels.•Pollutants were bioavailable and produced quick responses.•The untreated outfall discharge was more harmful than the ...treated one.•Up-regulation of Vtg transcription indicated presence of xenoestrogenic compounds in sewage.
To assess effects of urban discharges, biomarkers were measured in caged mussels in northern Iberian Peninsula. Lysosomal membrane stability and histopathology of gonad and digestive gland were analysed as general effect biomarkers. Exposure to specific pollutants was evaluated by autometallographical detection of metals, peroxisomal acyl-CoA oxidase activity, micronucleus test and transcription levels of vitellogenin and MT20 genes. Health status of mussels was impaired after 3days of caging at the untreated outfall discharge and at the waste water treatment plant effluent discharge to the estuary. The most relevant finding was the significant up-regulation of vitellogenin gene transcription in male mussels exposed to the untreated outfall discharge. Metals and xenoestrogenic endocrine disruptors were bioavailable in some discharges and disturbed the health status of mussels. Biomarkers were effective in the assessment of effects of urban discharges and could be implemented in operative controls required to assess the risks associated to effluent discharges.
Biomarkers are useful tools to study the health of estuarine and marine ecosystems. Biomarkers can be measured in different organisms, but mussels have acquired a global importance as sentinels in ...marine pollution-monitoring programs. In the present work, we aimed to determine the effects of different endocrine disruptors in mussels by using peroxisome proliferation as a biomarker of exposure to organic pollutants and the levels of vitellogenin (Vtg)-like proteins as biomarker of endocrine disruption. In experiment 1, mussels Mytilus edulis were exposed for 3 weeks to North Sea crude oil (NSO 0.5 ppm) and a mixture of 0.5 ppm NSO, 0.1 ppm alkylphenol mix, and 0.1 ppm extra polycyclic aromatic hydrocarbons (PAHs) (MIX). In experiment 2, mussels were exposed for 3 weeks to diallylphthalate (DAP 50 ppb), bisphenol-A (BPA 50 ppb), and tetrabromodiphenylether (TBDE 5 ppb). Peroxisome proliferation was assessed by measuring acyl-CoA oxidase (AOX) activity and peroxisomal volume density (VVp) in digestive gland. Vtg-like protein levels were measured in gonads by the alkali-labile phosphate (ALP) method. Gonad was also analyzed histologically, and the gonad index (GI) calculated. Mussels exposed to NSO and MIX showed significantly increased AOX activities and VVP compared with control animals. Significantly higher VVP was also found in DAP- and TBDE-exposed mussels. Effects on ALP and GI depended significantly on sex and time of year. In female mussels, ALP levels and GI were lower in the NSO group. In male mussels, ALP levels were significantly increased in the MIX group. The volume density of athretic oocytes was higher in the NSO and MIX exposure groups than in controls, and gonad resorption was observed in the BPA exposure group. Our results confirm the usefulness of peroxisome proliferation as a biomarker of exposure to organic contaminants in mussels and indicate that changes in Vtg-like proteins could be used as potential indicator of pollutant effects on mussel reproduction.
In aquatic organisms inhabiting polluted waters genes are activated to build an adaptive/compensatory defence against the possible effects of pollutants. Such responses can be used as biomarkers of ...exposure to chemical compounds, outlining the molecular mechanisms activated under specific pollution scenarios. With the aim of exploiting such approach in environmental health assessment, toxicologically relevant gene fragments were sequenced in the thicklip grey mullet (Chelon labrosus) and a toxicologically tailored low-density (160 genes) oligonucleotide microarray was customised. The tool was validated comparing organ/sex specific gene expression profiles and characterising responses under laboratory exposure to model chemicals. Finally, juvenile mullets were caged in a polluted harbour and hepatic gene expression profiles analysed after 5 and 21 days of deployment. Cages were deployed in the inner (IH) and outer (OH) Pasaia harbour, Bay of Biscay. Mussels (Mytilus galloprovincialis) were also caged as biological matrix for chemical bioaccumulation analysis and stress biomarkers measurements. Slightly higher concentrations of chemicals (metals, tributyltin, PAHs, phthalates) were quantified in IH than in OH, fish bile metabolites also revealing higher availability of PAHs in IH. Lysosome membrane stability in mussels was reduced, indicating stress condition in both sites. The developed microarray discriminated mullets showing distinctive expression profiles depending on site and deployment time. Genes related to immune and hypoxia responses were regulated comparing IH and OH at day 5. Phase I and II biotransformation genes, such as cyp2, cyp3 and ugt, were up-regulated in IH, together with the aryl hydrocarbon receptor 2 (ahr2) and the ahr repressor. Similarly, TBT-binding proteins and genes involved in lipid metabolism (pparγ, cyp7) were up-regulated with deployment time. Even if nowadays higher throughput approaches for gene expression analyses are available, the developed mullet tool constitutes a comprehensive tool to assess molecular responses of mullets exposed to pollutants, although it remains to be explored whether it can be applied to assess pollutant exposure in active pollution monitorings and in environmental health assessment.
•A toxicologically tailored low density microarray has been developed for thicklip grey mullet (C. labrosus).•The tool was validated comparing DEGs across tissues and under exposure to model chemicals.•Gene expression profiles distinguished mullets caged in two harbour areas with different chemical burdens.•Mullets in the most polluted site showed DEGs showing regulation of lipid metabolism, immune and hypoxia response pathways.
