Frank N.A.M. van Pelt

Variability of heat shock proteins and glutathione S-transferase in gill and digestive gland of blue mussel, Mytilus edulis

Glutathione S-transferase (GST) and heat shock proteins (hsps) 40, 60, 70 and 90 were determined by immunoblotting using actin as an internal control in Mytilus edulis from one station outside (site1) and three stations within (sites 2-4) Cork Harbour, Ireland. Comparisons were made between gill and digestive gland and between sites. Gill shows generally higher hsp 60, 70 and 90 while digestive gland has higher hsp 40. Site 1 showed higher gill hsps 40 and 70 than sites 2-4 while gill GST was higher in sites 3 and 4 than 1 and 2. Comparison with sites in the North Sea (site 5: outside Tjärnö in The Koster archipelago in the Skagerack) and Baltic Sea (site 6: Askö island) also revealed lower hsps 40 and 70 in site 6 (low salinity) than site 5 (high salinity) although hsps 60, 70 and 90 were detectable in digestive gland unlike sites 1-4. Previously, only hsp 70 had been studied at these sites [Mar. Environ. Res. 39. (1995), 181]. At the mRNA level, gill hsp 70 is 80-fold higher at Tjärnö than Askö. These data suggest that, while salinity may slightly decrease hsp 40 and 70, both hsp 70 and GST are selectively up-regulated by approx. 10- and 3-fold, respectively, at Tjärnö compared to the other sites which we attribute to exposure to more widely fluctuating pollution levels.

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.

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.

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.

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.

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.

Tipping the balance towards tolerance: the basis for therapeutic immune modulation by gold?

Gold salts have long been used in the treatment of rheumatoid arthritis. However, the basis for their therapeutic immune-modulating properties has never been satisfactorily explained. Furthermore, treatments are often marred by the development of adverse immune reactions such as hypersensitivity and even exacerbation of autoimmunity. We would like to propose a hypothesis to explain the basis for both the beneficial and adverse immune-modulating effects of gold in the treatment of rheumatoid arthritis. If accepted, this hypothesis will allow for the development of safer and more effective treatments with gold salts. The principle underlying this hypothesis also has broader implications for how immune hypersensitivity and tolerance are perceived.

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.

Acute and Chronic Liver Diseases Induced by Drugs or Xenobiotics

The incidence of adverse drug reactions to any given drug may be relatively low, but the total clinical impact of adverse drug reactions is actually substantial because of the number of drugs used and the number of patients treated. It has been estimated that around 7% of patients experience serious adverse drug reactions and that adverse drug reactions are the 4th to 6th leading cause of death (1). Hundreds of drugs and chemicals have been associated with hepatotoxic effects (1, 2, 3, 4, 5), and drugs are the most common cause of acute liver failure in the United States and Europe (6,7). In addition, serious druginduced hepatotoxicity has become one of the most frequent causes of postmarketing withdrawal, labeling changes, and restriction in use of medications (Table 1) (8, 9, 10).