Adsorption, uptake and toxicity of micro- and nanoplastics: Effects on terrestrial plants and aquatic macrophytes.

Plastic pollution is a new, pressing, environmental topic. Microplastics are considered contaminants of emerging concern and, consequently, microplastic research has grown exponentially in the last decade. Here, current knowledge regarding the impacts of micro- and nanoplastics on terrestrial plants and aquatic macrophytes is discussed, with a special focus on adsorption, uptake and toxicological effects. Our review reveals that a range of plants and macrophytes can adsorb or internalise plastic particles. Both processes depend on particle characteristics such as size and charge, as well as plant features including a sticky or hydrophobic surface layer. This finding is of concern given that plants and aquatic macrophytes are at the bottom of food webs and are a crucial component of the human diet. Therefore, there is a critical need for improved understanding of adsorption, uptake and impacts of micro- and nanoplastics, and the consequences thereof for trophic transfer, food safety and security. Also, a range of stress responses have been observed for many plant and macrophyte species after both short and long-term exposures to plastic particles. Given that some plastic particles can affect plant productivity, we surmise that plastic particles may potentially impact ecosystem productivity and function. Here we present a synthesis and a critical evaluation of the state of knowledge of micro- and nanoplastics and plants and macrophytes, identifying key questions for future research.

Beyond plastic microbeads - Short-term feeding of cellulose and polyester microfibers to the freshwater amphipod Gammarus duebeni.

Monitoring studies have revealed the presence of large numbers of natural as well as anthropogenic microfibers, plastic and non-plastic, in environmental samples. However, the interaction of organisms with microfibers is largely understudied. This is the first ecotoxicological study that compares short-term feeding of anthropogenic plastic and non-plastic microfibers on a consumer (leaf-shredding detritivores) species. The freshwater amphipod Gammarus duebeni was selected for this study as it is a model ecotoxicological species. After a 96-hour exposure, 58.3% and 41.7% of the amphipods contained cellulose or polyester fibers in their digestive tracts, respectively. Microfiber ingestion was analysed per polymers in presence or absence of food. The G. duebeni group exposed to 'polyester fibers in presence of food' accumulated highest numbers of microfibers in their digestive tracts (5.2 ±â€¯3.4 MFs/amphipod) followed by those exposed to 'cellulose in presence of food' (2.5 ±â€¯0.9 MFs/amphipod). A significantly (Three-way ANOVA, p-value <0.05) higher number of microfibers was found in the midgut-hindgut (posterior) sections, compared to the foregut (anterior) section. Microfiber uptake had no apparent short-term negative effect on amphipod survival at 96 h. Yet, as amphipods are both predators and prey, and therefore are key species in the aquatic food web, the rapid accumulation of anthropogenic microfibers in their digestive system has potentially further ecological implications. Future studies need to consider the possible transfer of ingested anthropogenic microfibers to higher trophic levels in freshwater communities.

Rapid fragmentation of microplastics by the freshwater amphipod Gammarus duebeni (Lillj.).

Microplastics have become ubiquitous in all environments. Yet, their environmental fate is still largely unknown. Plastic fragmentation is a key component of plastic degradation, which is mostly caused by abiotic processes over prolonged time scales. Here, it is shown that the freshwater amphipod Gammarus duebeni can rapidly fragment polyethylene microplastics, resulting in the formation of differently shaped and sized plastic fragments, including nanoplastics. Fragments comprised 65.7% of all observed microplastic particles accumulated in digestive tracts. Higher numbers of fragments were found in response to longer exposure times and/or higher microplastic concentrations. Furthermore, the proportion of smaller plastic fragments was highest when food was present during the depuration process. It is concluded that G. duebeni can rapidly fragment polyethylene microplastics and that this is closely associated with the feeding process. These results highlight the crucial role, currently understudied, that biota may play in determining the fate of microplastics in aquatic ecosystems.

Wood ash residue causes a mixture of growth promotion and toxicity in Lemna minor.

The use of wood as a sustainable biofuel results in the generation of residual wood ash. The ash contains high amounts of plant macronutrients such as phosphorus, potassium, calcium as well as several micronutrients. To explore the potential use of wood ash as a fertiliser, the growth enhancing properties of Sitka spruce (Picea sitchensis Bong.) wood ash were contrasted with the potential toxic action, using common duckweed (Lemna minor L.) as a model test species. The growth of L. minor exposed to wood bottom and fly ash solids and corresponding leachates was assessed in ultra-oligotrophic and eutrophic media. Ash solids and leachates were also tested as neutralized preparations. Suspended ash solids promoted L. minor growth up to concentrations of 2.5-5g/L. Leachates promoted growth up to 10g ash equivalents per litre, but for bottom ash only. Beneficial effects of wood ash were most pronounced on ultra-oligotrophic medium. However, on such nutrient-deficient medium severe inhibition of L. minor biomass and frond growth was observed at relatively low concentrations of fly ash (EC50=14g/L). On standard, eutrophic medium, higher concentrations of fly ash (EC50=21g/L), or neutralized fly ash (EC50=37g/L) were required to impede growth. Bottom ash, or neutralized bottom ash retarded growth at concentrations of 51g/L and 74g/L (EC50), respectively, in eutrophic medium. It appears that phytotoxicity is due to the elemental composition of the ash, its alkaline character, and possible interactions between these two properties. Growth promotion was due to the substantial content of plant nutrients. This study underlines the importance of the receiving environment (nutrient status and pH) in determining the balance between toxicity and growth promotion, and shows that the margin between growth promoting and toxicity inducing concentrations can be enlarged through ash neutralization.

Investigation of the genotoxic potential of the marine biotoxins azaspiracid 1-3.

Azaspiracids (AZAs) are the most recently discovered group of biotoxins and are the cause of azaspiracid shellfish poisoning (AZP) in humans. To date over thirty analogues have been identified. However, toxicological studies of AZAs are limited due to the lack of availability of toxins and toxin standards. Most data available are on acute toxicity and there are no data available on genotoxicity of AZAs. This study presents an integrated approach investigating the genotoxic potential of AZA1-3 in cell culture systems using the Comet assay combined with assays to provide information on possible apoptotic processes, cytotoxicity and changes in cell number. Results demonstrate a time and dose dependent increase in DNA fragmentation in most cell lines, indicating a genotoxic effect of AZA1-3. However, a significant reduction in cell number and a clear shift from early to late apoptosis was observed for all analogues in Jurkat T cells and HepG-2 cells; CaCo-2 cells did not show a clear apoptotic profile. Late apoptotic/necrotic cells correlate well with the percentage of tail DNA for all analogues in all three cell lines. All data taken together indicate that AZA1-3 is not genotoxic per se and demonstrate apoptotic/necrotic processes to be involved to some extent in AZAs toxicity. The sensitivities of cell lines and the different potencies of AZA1-3 are in agreement with the literature available. The order of sensitivity for all three AZAs tested in the present study is, in increasing order, CaCo-2 cells < HepG-2 cells < Jurkat T cells. The order of potency of AZA1-3 varies among the cell lines.

Assessment of emerging biotoxins (pinnatoxin G and spirolides) at Europe's first marine reserve: Lough Hyne.

Active and passive sampling methods were employed over a four-month period, at a site off the South-West coast of Ireland, to characterise the occurrence of cyclic imines in the water column. The marine toxins 13-desmethyl-SPXC, 20-methyl SPXG toxins and pinnatoxin G were detected using active sampling from Diaion HP-20 resin. Seven water depths were sampled to determine stratification of the toxins in the water column using Solid Phase Adsorption and Toxin Tracking (SPATT). Both 13-desmethyl-SPXC and pinnatoxin G were detected using two different resin types; Diaion HP-20 and Amberlite XAD761. HP-20 proved more effective at accumulating the toxins, with a higher percentage of positive samples and a higher ratio of toxin adsorbed relative to XAD761. No temporal variation in toxin-quantities was detected, indicating that there was no change in density of causative algal species in the water column. Pinnatoxin G was detected more frequently from surface to 30 m depth, with a similar pattern observed for 13-desmethyl-SPXC occurrence using XAD761. No difference in the occurrence of 13-desmethyl-SPXC was observed between depths using HP-20 resin. This is the first reported incidence of pinnatoxin G in Irish waters and highlights cyclic imines as emerging toxins in European waters.

Does the marine biotoxin okadaic acid cause DNA fragmentation in the blue mussel and the pacific oyster?

Two bivalve species of global economic importance: the blue mussel, Mytilus edulis and the pacific oyster, Crassostrea gigas were exposed in vivo, to the diarrhoetic shellfish toxin okadaic acid (OA), and impacts on DNA fragmentation were measured. Shellfish were exposed using two different regimes, the first was a single (24 h) exposure of 2.5 nM OA (∼0.1 µg/shellfish) and algal feed at the beginning of the trial (T0), after which shellfish were only fed algae. The second was daily exposure of shellfish to two different concentrations of OA mixed with the algal feed over 7 days; 1.2 nM OA (∼0.05 µg OA/shellfish/day) and 50 nM OA (∼2 µg OA/shellfish/day). Haemolymph and hepatopancreas cells were extracted following 1, 3 and 7 days exposure. Cell viability was measured using the trypan blue exclusion assay and remained above 85% for both cell types. DNA fragmentation was examined using the single-cell gel electrophoresis (comet) assay. A significant increase in DNA fragmentation was observed in the two cell types from both species relative to the controls. This increase was greater in the pacific oyster at the higher toxin concentration. However, there was no difference in the proportion of damage measured between the two cell types, and a classic dose response was not observed, increasing toxin concentration did not correspond to increased DNA fragmentation.

Application of passive (SPATT) and active sampling methods in the profiling and monitoring of marine biotoxins.

Solid phase adsorbent and toxin tracking (SPATT) enables temporally and spatially integrated monitoring of biotoxins in aquatic environments. Monitoring using two adsorbent resins was performed over a four-month period at Lough Hyne Marine Reserve, Ireland. A range of Diarhettic Shellfish Poisoning (DSP) toxins were detected from SPATT extracts throughout the study period. The majority of biotoxins were detected in the top 20-30 m of the water column and a spike in toxin accumulation was measured during August 2010. Phytoplankton analysis confirmed the presence of toxin-producing species Dinophysis acuta and Dinophysis acuminata during the bloom. SPATT has the potential to provide useful information on phycotoxin distribution in the water column; enabling evidence-based decisions regarding appropriate depths for obtaining phytoplankton and shellfish samples in marine biotoxin monitoring programmes. Active sampling was performed continuously over 7-days and high quantities of toxins were successfully accumulated in the HP-20 resin, okadaic acid (∼13 mg), dinophysis toxin-2 (∼29 mg), pectenotoxin-2 (∼20 mg) and pectenotoxin-2-seco acid (∼6 mg) proving this an effective method for accumulating DSP toxins from the marine environment. The method has potential application as a tool for assessing toxin profiles at proposed shellfish harvesting sites.

Elucidation of the mass fragmentation pathways of the polyether marine toxins, dinophysistoxins, and identification of isomer discrimination processes.

RATIONALE: Most of the liquid chromatography/mass spectrometry (LC/MS) methods that have been developed for the analysis of Diarrhetic Shellfish Poisoning (DSP) toxins in shellfish and algae samples have been unable to differentiate the isomers okadaic acid (OA) and dinophysistoxin-2 (DTX2), unless separated by chromatography. Since there are many bioconversion products of these compounds it is imperative to determine characteristic product ions, which can provide unequivocal identification of OA and DTX2 and their analogs. METHODS: Using electrospray ionization, the fragmentation processes for two types of precursor ions, [M+Na](+) and [M-H](-), of the polyether marine toxins, dinophysistoxins (DTXs), were studied using a hybrid linear ion trap Orbitrap mass spectrometer which provided high mass accuracy data in combination with multiple tandem mass (MS(n)) spectra. Three structurally related toxins were compared; okadaic acid (OA), dinophysistoxin-2 (DTX2) and dinophysistoxin-1 (DTX1). A quick multiple reaction monitoring (MRM) LC/MS/MS method was developed utilizing the characteristic precursor/product ion mass transitions. RESULTS: Comparison of the high-resolution product ion, [M-H](-), spectra of these toxins featured dominant signals that resulted from two six-centered rearrangements and previously proposed fragmentation pathways for the ion of m/z 321 and 293 have been corrected and identified. By contrast, the [M+Na](+) product ion spectra only revealed distinctive ions for the isomers, OA (m/z 595, 443 and 151) and DTX2 (m/z 581, 429 and 165). To illustrate the benefits of this study, a mass selective LC/MS/MS method was developed in which the isomers OA and DTX2 co-eluted but were distinguished using the mass transitions, m/z 827/595, 827/443 (OA) and m/z 827/581, 827/429 (DTX2). CONCLUSIONS: Comparison of OA, DTX2 and DTX1 led to the correction of proposed negative ion mode fragmentation pathways. Through extensive study and comparison of the [M+Na](+) product ion spectra, distinctive product ions were identified which allowed for these compounds to be identified and distinguished without separation for the first time.

Alumina nanoparticles enhance growth of Lemna minor.

The industrial use of nanoparticles is rapidly increasing, and this has given rise to concerns about potential biological impacts of engineered particles released into the environment. So far, relatively little is known about uptake, accumulation and responses to engineered nanoparticles by plants. In this study, the effects of alumina nanoparticles on growth, morphology and photosynthesis of Lemna minor were quantified. It was found that alumina nanoparticles substantially increase biomass accumulation of L. minor. Such a stimulatory effect of alumina nanoparticles on growth has not been reported previously. Enhanced biomass accumulation was paralleled by morphological adjustments such as increased root length and number of fronds per colony, and by increased photosynthetic efficiency. Metal nanoparticles have previously been shown to enhance the energy transfer efficiency of isolated reaction centres; therefore it is proposed that the mechanism underlying the alumina mediated enhancement of biomass accumulation in L. minor is associated with increased efficiencies in the light reactions of photosynthesis.

Mussels increase xenobiotic (azaspiracid) toxicity using a unique bioconversion mechanism.

Azaspiracid Poisoning (AZP) is a human toxic syndrome which is associated with the consumption of bivalve shellfish. Unlike other shellfish, mussels contain a large array of azaspiracid analogs, many of which are suspected bioconversion products. These studies were conducted to elucidate the metabolic pathways of azaspiracid (AZA1) in the blue mussel (Mytilus edulis) and revealed that the main biotransformation product was the more toxic demethyl analog, AZA3. To elucidate the mechanism of this C-demethylation, an unprecedented xenobiotic bioconversion step in shellfish, AZA1 was fed to mussels that contained no detectable azaspiracids. Triple quadrupole mass spectrometry (MS) and high resolution Orbitrap MS were used to determine the uptake of AZA1 and the toxin profiles in three tissue compartments of mussels. The second most abundant bioconversion product was identified as AZA17, a carboxyl analog of AZA3, which is a key intermediate in the formation of AZA3. Also, two pairs of isomeric hydroxyl analogs, AZA4/AZA5 and AZA7/AZA8, have been confirmed as bioconversion products for the first time. Ultra high resolution (100 k) MS studies showed that the most probable structural assignment for AZA17 is 22-carboxy-AZA3 and a mechanism for its facile decarboxylation to form AZA3 has been proposed.

Food contaminant analysis at ultra-high mass resolution: application of hybrid linear ion trap - orbitrap mass spectrometry for the determination of the polyether toxins, azaspiracids, in shellfish.

The biotoxins, azaspiracids (AZAs), from marine phytoplankton accumulate in shellfish and affect human health by causing severe gastrointestinal disturbance, diarrhea, nausea and vomiting. Specific and sensitive methods have been developed and validated for the determination of the most commonly occurring azaspiracid analogs. An LTQ Orbitrap mass spectrometer is a hybrid instrument that combines linear ion trap (LIT) mass spectrometry (MS) with high-resolution Fourier transform (FT) MS and this was exploited to perform simultaneous ultra-high-resolution full-scan MS analysis and collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Using the highest mass resolution setting (100,000 FWHM) in full-scan mode, the methodology was validated for the determination of six AZAs in mussel (Mytilus galloprovincialis) tissue extracts. Ultra-high mass resolution, together with a narrow mass tolerance window of ±2 mDa, dramatically improved detection sensitivity. In addition to employing chromatographic resolution to distinguish between the isomeric azaspiracid analogs, AZA1/AZA6 and AZA4/AZA5, higher energy collisionally induced dissociation (HCD) fragmentation on selected precursor ions were performed in parallel with full-scan FTMS. Using HCD MS/MS, most precursor and product ion masses were determined within 1 ppm of the theoretical m/z values throughout the mass spectral range and this enhanced the reliability of analyte identity.For the analysis of mussels (M. galloprovincialis), the method limit of quantitation (LOQ) was 0.010 µg/g using full-scan FTMS and this was comparable with the LOQ (0.007 µg/g) using CID MS/MS. The repeatability data were; intra-day RSD% (1.8-4.4%; n = 6) and inter-day RSD% (4.7-8.6%; n = 3). Application of these methods to the analysis of mussels (M. edulis) that were naturally contaminated with azaspiracids, using high-resolution full-scan Orbitrap MS and low-resolution CID MS/MS, produced equivalent quantitative data.

In vitro exposure of jurkat T-cells to industrially important organic solvents in binary combination: interaction analysis.

Humans are frequently exposed to mixtures of environmental pollutants at low levels over prolonged periods of time yet most toxicity studies deal with acute exposure to high concentrations of single chemicals. Investigation of the biological effects and possible toxic interactions during long-term exposure to such mixtures is warranted. Here Jurkat T-cells were exposed to toluene, n-hexane and methyl ethyl ketone in binary combination. Concentration ranges were centered on thresholds at which the individual agents caused cell toxicity under otherwise similar conditions, and concentrations were confirmed by headspace gas chromatography. After 5 days cells were harvested and toxicity measured in terms of membrane damage (lactate dehydrogenase [LDH] leakage), perturbations in [Ca(2+)](i) and changes in glutathione redox status. Data for all three endpoints were subjected to isobolographic analysis to test for interaction between components of the solvent mixture. Almost all combinations of toluene and n-hexane elicited greater than additive toxicity in terms of each of the three endpoints, as did methyl ethyl ketone (MEK)/n-hexane and MEK/toluene combinations for the LDH and glutathione endpoints. The main exceptions were the two combinations involving MEK, which caused less than additive effects on perturbations of [Ca(2+)](i). It is concluded that toxicity in immune-derived T cells may exhibit greater than additive effects when there is coexposure to organic solvents. This may have implications for risk assessment of environmental exposure to these agents.

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L.

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.

Sub-chronic toxicity of low concentrations of industrial volatile organic pollutants in vitro.

Organic solvents form an important class of pollutants in the ambient air and have been associated with neurotoxicity and immunotoxicity in humans. Here we investigated the biological effects of sub-chronic exposure to industrially important volatile organic solvents in vitro. Jurkat T cells were exposed to toluene, n-hexane and methyl ethyl ketone (MEK) individually for 5 days and solvent exposure levels were confirmed by headspace gas chromatography. A neuroblastoma cell line (SH-SY5Y) was exposed to toluene for the same period. Following exposure, cells were harvested and toxicity measured in terms of the following endpoints: membrane damage (LDH leakage), perturbations in intracellular free Ca(2+), changes in glutathione redox status and dual-phosphorylation of MAP kinases ERK1/2, JNK and p38. The results show that sub-chronic exposure to the volatile organic solvents causes membrane damage, increased intracellular free calcium and altered glutathione redox status in both cell lines. However, acute and sub-chronic solvent exposure did not result in MAP kinase phosphorylation. Toxicity of the solvents tested increased with hydrophobicity. The lowest-observed-adverse-effect-levels (LOAELs) measured in vitro were close to blood solvent concentrations reported for individuals exposed to the agents at levels at or below their individual threshold limit values (TLVs).

Validation of a method for acute and subchronic exposure of cells in vitro to volatile organic solvents.

In vitro assessment of organic solvents can be problematic as the volatile nature of these compounds makes maintaining a constant exposure level difficult. However, a stable exposure level must be maintained if reliable dose response data are to be obtained. Here we describe a gas-tight glass exposure system which allows prolonged exposure of cultured cells to constant concentrations of volatile organic solvents. The system permits convenient sampling of gas and liquid phases for reliable quantification of solvent concentration. We determined medium/air partition coefficients (K) for toluene, n-hexane and methyl ethyl ketone which can be used to calculate liquid phase solvent exposure levels in an in vitro system specifically designed for organic solvent exposure. Cultured cells were exposed to these compounds for five days and toxicity assessed by trypan blue exclusion. Headspace gas chromatography was used to determine K in RPMI-1640 and EMEM tissue culture medium at 37 degrees C. The presence of cells in the system at levels normally used in in vitro exposure systems did not significantly alter solvent partitioning. Equilibrium liquid phase solvent concentrations were measured by gas chromatography for two of the compounds to confirm that exposure levels calculated using K were correct. Results show that sub-chronic exposure to volatile organic solvents causes a dose dependent decrease in Jurkat T-cells and SH-SY5Y viability. Solvent potency increased with lipophilicity (n-hexane>toluene>MEK).

A two-species biomarker model for the assessment of sediment toxicity in the marine and estuarine environment using the comet assay.

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-associated contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to contaminants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph/blood, gill and digestive gland/liver from the clam Tapes semidecussatus and turbot Scophthalmus maximus, using the single cell gel electrophoresis (Comet Assay). Both animals were exposed for three weeks to sediment samples collected from a polluted site and a 'clean' reference site. The level of DNA damage was assessed using an image analysis package and expressed as % tail DNA. Throughout the study, significant differences in DNA damage were recorded for each tissue type, in both species, between animals exposed to the two sediment samples. However, turbot appeared to be a more sensitive indicator species, because, due to lower background levels, they were able to detect a significant difference between reference site and background values. This suggests that turbot, rather than clams, are more suitable as a sentinel species for the assessment of genotoxic impact of low-level contamination in aquatic sediments and highlights the need for a two- or multi-species approach.

Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay.

Immune modulation by heavy metals may cause serious adverse health effects in humans, although the mechanisms involved are not well understood. Both cadmium and lead are important environmental and occupational toxins. Therefore, in the current study, the costimulatory/adjuvant effects and the T-cell-activating potential of these metals (i.e., CdCl2 and PbCl2), are examined. These immune-modulating properties are critical in the development of conditions such as allergy, hypersensitivity, and autoimmunity. Using the direct popliteal lymph node assay (PLNA) and reporter antigen-popliteal lymph node assay (RA-PLNA) both metals were examined individually for immunotoxicity. Mercury (i.e., HgCl2) was included for comparative purposes as its effects in the RA-PLNA are well documented. Seven days following a single footpad injection containing metal and/or RA (trinitrophenyl-ovalbumin [TNP-OVA] or TNP-Ficoll), BALB/c mice were sacrificed and the popliteal lymph nodes (PLNs) removed. PLN cellularity, TNP-specific antibody-secreting cells (ASCs), and lymphocyte subsets were assessed. All three metals strongly stimulated T- and B-cell proliferation and ASC production following coinjection with the RA TNP-OVA. In each case, ASC production was skewed towards the IgG1 isotype. In addition, all three metals induced IgG production to TNP-Ficoll (although relatively weakly in the case of Cd). These results show that each of these metals can provide adjuvant signals to promote lymphocyte proliferation and enhance adaptive immune responses to unrelated antigens. Skewing of immune responses towards T helper type 2 responses suggests that each of these metals can enhance allergic and hypersensitivity reactions to environmental antigens. Furthermore, the induction of IgG responses to TNP-Ficoll, a T-cell-independent antigen, indicates that each of these metals can activate neoantigen-specific T cells. T-cell activation by metals can lead to metal hypersensitivity and has been implicated in the development of autoimmunity. This is the first report of immune modulation by CdCl2 and PbCl2 in the RA-PLNA.

Cellular responses in primary epidermal cultures from rainbow trout exposed to zinc chloride.

In this study, we examined the effects of zinc chloride (ZnCl(2)) (0-200mg/L) on primary epidermal cultures from Oncorhynchus mykiss. Increases in the rate and amount of mucus released were detected post-exposure, as was a dose-dependent increase in the synthesis of acidic glycoproteins. The cytotoxicity of ZnCl(2) to the cultures was significantly increased (P< or =0.05) when exposures were conducted in serum-free medium as opposed to medium containing serum. Significant increases in the levels of apoptosis and necrosis were observed with increasing exposure concentration, but these were seen to decrease over time. The in vitro cytological and pathological changes observed in this study were found to be in accordance with previously published in vivo studies on the effects of heavy metals on the integument. This model system may help to further elucidate the effects of ecotoxicants on the external innate immune system of fish.

Protein carbonylation and heat shock response in Ruditapes decussatus following p,p'-dichlorodiphenyldichloroethylene (DDE) exposure: a proteomic approach reveals that DDE causes oxidative stress.

Protein carbonylation and levels of heat shock proteins (hsp; 60, 70 and 90 kDa) were measured in gill, mantle and digestive gland of Ruditapes decussatus following exposure to p,p'-dichlorodiphenyldichloroethylene (DDE). Heat shock response was measured by immunoblotting using antibodies specific to heat shock proteins (hsps). Densitometry analysis of individual bands revealed no difference between control and treated samples except appearance of hsp90 in DDE-treated mantle. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and two-dimensional electrophoresis coupled with western blotting. Immunoblotting with dinitrophenol-specific antibody revealed extensive differences in both extent and number of carbonylated proteins in mantle and digestive gland in response to DDE while gill was unaffected. These results demonstrate for the first time that DDE causes tissue-specific formation of reactive oxygen species in clams.