Extraction and cleaning methods to detect yessotoxins in contaminated mussels.

Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.

[Natriuretic peptides in heart failure]. / Peptidi natriuretici come guida alla prognosi e terapia nello scompenso cardiaco.

The recent discovery of cardiac endocrine function, together with the development of accurate and feasible assay methods for cardiac natriuretic hormone evaluation, i.e. for B-type natriuretic peptide (BNP) and inactive peptide NT-proBNP have confirmed their pathophysiological and clinical significance for cardiovascular disease assessment. Concerning heart failure, their value is for diagnostic screening in selected/unselected populations, for differential diagnosis of dyspnea and for prognostic stratification, and as a guide for follow-up and treatment of patients. Recent Italian recommendations pointed out that BNP/NT-proBNP has a role in ruling-out the diagnosis of heart failure in patients with dubious signs/symptoms: plasma BNP/NT-proBNP concentrations help in the clinical evaluation of chronic heart failure patients when risk stratification is needed, whereas the routine BNP/NT-proBNP assay is still not recommended to guide therapeutic decision-making.

Swiss mice CD1 fed on mussels contaminated by okadaic acid and yessotoxins: effects on thymus and spleen.

The toxicity of okadaic acid (OA) and yessotoxins (YTXs) was studied in mice orally fed on (i) OA (17.80+/-2.41 microg/kg) for 24 h and mouse feed for 24 h; (ii) OA (17.2+/-2.13 microg/kg) plus YTXs (1.30+/-0.12 mg/kg) for 24 h and mouse feed for 24 h; (iii) OA (18.88+/-1.86 microg/kg) plus YTXs (1.45+/-0.12 mg/kg) for 24 h. After toxin treatments the thymus and spleen were examined. More severe morpho-functional modifications were found in the thymus, which presented atrophy, a significant depletion in the lymphoid compartment and angiogenesis. In spite of the impairment, a number of inflammatory cells, reactive to anti-cytokine antibodies, were recruited. Moreover, greater expression of matrix metalloproteinase-9, particularly in cells located near new blood vessels, was observed. Thymus injury was still observed after 48 h. Histopathological changes to the spleen were more evident in mice orally treated for 24 h and immediately sacrificed. The organ showed a significant loss of volume and a fibrous component invaded regions involved in immune functions. In white pulp the marginal zones were reduced, lymphoid nodules contained large germinal centres and the periarteriolar lymphoid sheaths showed cellular depletion. An inflammatory cell response was activated by the recruitment of granulocytes, an increased number of active macrophages and increased immunoreactivity to cytokines. Unlike in the thymus, some evidence of recovery was seen in the spleen. The data suggest that low oral doses of OA alone or OA plus YTXs are able to provoke immunostimulation and systemic immunotoxicity, thus also indicative of tumorigenic properties.

Lethal and sub-lethal yessotoxin dose-induced morpho-functional alterations in intraperitoneal injected Swiss CD1 mice.

Histological and immunocytochemical investigations were performed on different organs (brain, duodenum and thymus) of mice following lethal (420 microg/kg) or sublethal (10 microg/kg) intraperitoneal injection of yessotoxin (YTX). No morpho-functional modifications were observed in large neurons of the cerebral and cerebellar cortex with the sub-lethal dose, nor in the cerebral cortex with the lethal dose. The duodenum also did not show significant alterations. However, there was an inflammation response to the toxin, in which blood cells and cytokines were involved. This was more evident with the lethal YTX dose. The thymus and, in general, the immune system are the main targets of YTX at both the concentrations used. Furthermore, the alterations present in the thymus may support tumorigenic implications.

Acute toxic effect of the algal yessotoxin on Purkinje cells from the cerebellum of Swiss CD1 mice.

Swiss CD1 mice died less than 2 h after intraperitoneal injection of 420 microg/kg of algal yessotoxin (YTX). The morphological, histochemical and immunocytochemical studies performed on the cerebellar cortex revealed damage to the Purkinje cells. The main cytological alterations were observed in the cytoplasm, while less sufferance was detected in the nucleus. The immunocytochemical experiments showed an increased positivity to S100 protein while there was a decreased response to calbindin D-28K, beta-tubulin and neurofilaments. These changes in intracellular Ca(2+)-binding proteins and the modifications in the cytoskeletal components of Purkinje cells suggest that YTX may be involved in neurological disorders.

Algal biotoxins of marine origin: new indications from the European Union.

Marine biotoxins, more or less complex molecules with various origins that can accumulate in the tissues of fish products through the food chain, are reviewed. The EU, aware of the danger incurred in eating certain fish products, has issued a set of hygiene and health directives for the purpose of preventing disease and safeguarding consumer health. In particular, directive 91/492/EEC, of 15 July 1991, lays down the sanitary norms applicable to the production and commercialization of live bivalve molluscs, echinoderms, tunicates and marine gastropods and regulates the whole system involving these products from their origin to consumption. More recently, through Commission Decision dated 15 March 2002 (EC OJ 175/62 of 16.3.2002) the EU has set new standards for the implementation of directive 91/492/EEC with respect to the maximum levels and analysis methods for some marine biotoxins.

Immunolocalization of yessotoxins in the mussel Mytilus galloprovincialis.

This study reports, for the first time, immunocytochemical evidence of the distribution of algal yessotoxins (YTXs) in the mussel Mytilus galloprovincialis. Immunopositivity to YTXs was found in immunocytes and in the digestive gland. With regards the gland, the positivity was mainly present in the lumen of both tubules and ducts. No YTXs were detected in the gonads, while the presence of toxins in the gills cannot be excluded. The data are supported by both HPLC analysis and functional assays.

Structural elucidation of a new cytotoxin isolated from mussels of the Adriatic sea.

A detailed analysis of the toxic composition in the hepatopancreas of mussels from northern Adriatic sea has been performed. Along with some polyether toxins of DSP (diarrhetic shellfish poisoning) type, such as yessotoxin and its analogues, which are responsible for a variety of human seafood poisonings throughout the world, we have now isolated a new type of toxin, the chlorosulfolipid 1, which is completely different in structure from the polyether DSP-toxins isolated so far. The structural determination of the new toxin, including its absolute stereochemistry, has been performed by extensive NMR analysis and molecular mechanics and dynamics calculations.

42,43,44,45,46,47,55-Heptanor-41-oxohomoyessotoxin, a new biotoxin from mussels of the northern Adriatic sea.

The diarrhetic shellfish toxin composition in the digestive glands of mussels from the northern Adriatic sea was investigated. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, 42,43,44,45,46,47,55-heptanor-41-oxohomoyessotoxin, 1. Its structure was determined by (1)H NMR spectroscopy and mass spectrometry.

Structure determination of carboxyhomoyessotoxin, a new yessotoxin analogue isolated from adriatic mussels.

The contamination of shellfish with marine biotoxins derived from microalgae represents a serious problem for shellfish industries and public health. This study investigated the composition of diarrhetic shellfish toxins in the digestive glands of mussels from the Northern Adriatic Sea. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, carboxyhomoyessotoxin, whose structure was determined by mass spectrometry and (1)H NMR spectroscopy.

High sensitivity bioassay of paralytic (PSP) and amnesic (ASP) algal toxins based on the fluorimetric detection of [Ca(2+)](i) in rat cortical primary cultures.

A high sensitivity bioassay able to recognise small amounts of paralytic and amnesic toxins in algal acetic extracts is described. The method is based on the measure of intracellular [Ca(2+)](i) in primary cultures of rat cortical neurones preloaded with Fura-2 and submitted to electrical field stimulation. Under normal conditions the basal [Ca(2+)](i) level was about 50-100 nM and was nearly doubled during the peaks induced by trains of electrical pulses at 10 Hz for 10 s. Saxitoxin (STX) 3.5 nM and tetrodotoxin (TTX) 24 nM halved the peaks height without affecting basal [Ca(2+)](i). Conversely, domoic acid increased the basal [Ca(2+)](i) (EC(50)=3. 7 microM) and decreased the calcium peaks (EC(50)=7.3 microM). CNQX (a competitive antagonist of AMPA/KA receptors) at 10 microM shifted to the right by a factor of 3 the concentration-response curves of domoic acid. The extracts of non-toxic algae were well tolerated by up to 10 microg protein/ml, whereas extracts of Alexandrium lusitanicum at 1-4 microg protein/ml reduced [Ca(2+)](i) peaks and increased basal calcium levels. This toxic effect of A. lusitanicum was unexpected since parallel HPLC analysis showed only the presence of gonyautoxins, known to act like saxitoxin. Therefore, the bioassay on rat cortical neurones revealed a complex composition of the toxins present in A. lusitanicum. The relevance of fluorimetric detection of [Ca(2+)](i) in primary neuronal cultures in the evaluation of algal risk is stressed.

High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea.

Identification of YTX and homoYTX in natural phytoplankton populations containing significant amounts of Gonyaulax polyedra and determination of detailed toxin profiles of mussels (Mytilus galloprovincialis) periodically collected from two sites of the Northern Adriatic coast from February to October 1997 was performed by LC-FLD following derivatization with ADAM or DMEQ-TAD and LC-MS and LC-MS-MS. OA and YTX concentrations were recorded in the range 0.11-2.31 and 0.18-9.02 microg per g of hepatopancreas, respectively. HomoYTX was also detected both in phytoplankton and mussel samples.

Isolation of 45-hydroxyyessotoxin from mussels of the Adriatic Sea.

The diarrhetic shellfish toxin composition in the hepatopancreas of mussels from the northern Adriatic sea was investigated. The major toxins were shown to be yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxyyessotoxin (45-OHYTX), identified by comparison of their chromatographic and spectral properties with those reported in the literature.

Yessotoxin in mussels of the northern Adriatic Sea.

This study investigated the composition of diarrhoetic shellfish toxins in the hepatopancreas of mussels from the northern Adriatic Sea. The major toxins were shown to be yessotoxin, identified by its chromatographic properties and spectral data, and okadaic acid, detected both by fluorometric high-performance liquid chromatography and by comparison of its spectral properties with those of an authentic sample.

First report of pectenotoxin-2 (PTX-2) in algae (Dinophysis fortii) related to seafood poisoning in Europe.

Pectenotoxin-2 (PTX-2), a polyether-lactone included in the neutral class of diarrhoetic shellfish poisoning (DSP) toxins, has been unambiguously detected in Dinophysis fortii collected in the northern Adriatic Sea (Emilia Romagna coasts). This is the first report of such a toxin in Europe. This lipid soluble toxin was identified both in crude methanolic phytoplankton extract and in the neutral fraction obtained by extract chromatography on a basic alumina column. The techniques used were reversed phase high-performance liquid chromatography followed either by UV diode-array detection (LC-UV-DAD) or by mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS-MS) using an atmospheric-pressure ionization source and an ionspray interface. Okadaic acid (OA) was also found in the D. fortii specimens and quantified as 15 pg/cell. Although quantitation of PTX-2 was not possible due to the lack of pure toxin, the high PTX-2:OA ratio suggested PTX-2 was significant in the D. fortii specimens. The presence of PTX-2 in a region with no previous report of DSP neutral toxic compounds may indicate a risk of human poisoning. Serious efforts should therefore be made to develop suitable routine methods capable of detecting the presence of PTXs in biological materials of marine origin, in order to assure the wholesomeness of seafood products.

ASP, DSP, NSP and PSP monitoring in 'mucilaginous aggregates' and in mussels in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in 1988, 1989 and 1991.

In this study, monitoring of marine biotoxins in "mucilaginous aggregates" and in mussels from coastal area of Emilia Romagna (Northern Adriatic Sea) in June-August 1988, 1989 and 1991, are reported. Both "mucilaginous aggregates" and mussels were analysed for NSP and PSP in 1988, and ASP, DSP, NSP, PSP in 1989, 1991. Concerning "mucilaginous aggregate" any presence of biotoxins was never detected. In the mussels it was possible to exclude the presence of PSP, ASP and NSP, but very high levels of DSP were shown in all the considered periods, in relation to the presence in the sea water of cells of the Dinophysis genus.