Carmela Dell'Aversano

Complex palytoxin-like profile of Ostreopsis ovata. Identification of four new ovatoxins by high-resolution liquid chromatography/mass spectrometry.

Over the past decades, Italian coastlines have been plagued by recurring presence of the benthic dinoflagellate Ostreopsis ovata. Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous studies have confirmed the presence of ovatoxin-a (OVTX-a) as the major toxin of the algal toxin profile together with small amounts of putative palytoxin (PLTX). In our ongoing research on O. ovata toxins we report herein on in-depth investigation of an O. ovata culture carried out by high-resolution (HR) liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS(2)). Particularly, the presence of putative PLTX and OVTX-a was confirmed and the occurrence in the extract of four new palytoxin-like compounds, OVTX-b, -c, -d, and -e, was highlighted. Elemental formulae have been assigned to the new ovatoxins and information has been gained about their structural features. A quantitative study of the O. ovata culture extract indicated that the whole of the new ovatoxins represents about 46% of the total toxin content and, thus, their presence has to be taken into account when LC/MS-based monitoring programs of either plankton or contaminated seafood are carried out.

Comparative growth and toxin profile of cultured Ostreopsis ovata from the Tyrrhenian and Adriatic Seas.

Massive blooms of the benthic dinoflagellate Ostreopsis ovata Fukuyo have recently occurred along the whole Italian coastlines, both Tyrrhenian and Adriatic, resulting sometimes in benthonic biocenosis sufferings and, occasionally, in human health problems. In this work, two strains of O. ovata collected in 2006 along the Adriatic and Tyrrhenian coastlines and grown in culture were studied to characterize their growth and toxin profile. The two strains showed different cell volumes, the Adriatic strain being nearly twice bigger than the Tyrrhenian, but they had similar slow growth rates. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that both strains produce putative palytoxin (pPLTX) and ovatoxin-a (OVTX-a), a palytoxin-like compound presenting 2 oxygen atoms less than palytoxin. Toxin content was determined at the end of the stationary and exponential growth phases and reached the highest value in the Adriatic strain at the end of the stationary phase, with concentrations of 353.3 microg l(-1) for OVTX-a and 30.4 microg l(-1) for pPLTX. Toxin released in the growth medium was also measured and resulted to be the highest at the end of the stationary phase, suggesting that a long lasting bloom could enhance the toxin content in the water and cause toxic effects in people inhaling the aerosol.

Complex yessotoxins profile in Protoceratium reticulatum from north-western Adriatic sea revealed by LC-MS analysis.

While the occurrence of yessotoxin (YTX) has been reported worldwide from Protoceratium reticulatum, the biogenetic origin of some YTX analogues is still unknown, thus raising an issue whether they are metabolites of YTX formed in mussels or true products of different dinoflagellate species. Findings reported herein suggest that P. reticulatum from the north-western Adriatic sea is responsible for production, together with YTX (1), of homoYTX (2), 45-OHYTX (3), carboxyYTX (5), and noroxoYTX (7). YTX and its analogues have been determined by high performance liquid chromatography coupled with electrospray ion trap mass spectrometry (HPLC-MS and HPLC-MSn experiments). The result is the first to confirm production of these YTX analogues from this species and indicate it as a producing organism of homoYTX, 45-OHYTX, carboxyYTX, and noroxoYTX found in shellfish.

Stereostructure and Biological Activity of 42-Hydroxy-palytoxin: A New Palytoxin Analogue from Hawaiian Palythoa Subspecies

This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.

Complex toxin profile of Mytilus galloprovincialis from the Adriatic sea revealed by LC-MS.

This paper reports on the determination of toxin profile of mussels (Mytilus galloprovincialis) collected in November-December 2003 along the Emilia Romagna coasts (Italy) when a high concentration of Alexandrium ostenfeldii cells was detected in seawater. Detailed liquid chromatography-mass spectrometry (LC-MS) analyses were performed on the crude extracts in both selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. They revealed that M. galloprovincialis had accumulated the three major spirolides produced by the alga, namely 13-desMethyl spirolide C, 13,19-didesMethyl spirolide C and 27-hydroxy-13,19-didesMethyl spirolide C, which fully accounted for toxicity of lipophilic extracts shown in mouse bioassay. Interestingly, yessotoxin (YTX) and its analogues were still present in mussel polar extracts but YTX itself was not the major toxin contained in mussels. The presence of pectenotoxin-2 seco acid (PTX-2sa) and its putative epimer was also assessed. The presence of azaspiracids, never reported from the Adriatic sea, as well as of diarrhetic shellfish poisoning toxins (okadaic acid, dinophysistoxins and OA esters) and domoic acid, long known as contaminants of Adriatic mussels, was also investigated.

Chemistry of Verongida sponges. VII bromocompounds from the caribbean sponge Aplysina archeri

Abstract A detailed analysis of the secondary liposoluble metabolites of the Verongida sponge Aplysina archery has been performed. Ten bromotyrosine derivatives have been identified of which one, 1, is a novel compound. Structure of 1 has been assigned on the basis of spectroscopic evidence including 2D-NMR experiments. Absolute configuration has been suggested by helicity rule.

Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-κB

Background Palytoxin and, likely, its analogues produced by the dinoflagellate genus Ostreopsis, represent a class of non-proteinaceous compounds displaying high toxicity in animals. Owing to the wide distribution and the poisonous effects of these toxins in humans, their chemistry and mechanism of action have generated a growing scientific interest. Depending on the exposure route, palytoxin and its Ostreopsis analogues may cause several adverse effects on human health, including acute inflammatory reactions which seem more typical of cutaneous and inhalation contact. These observations have led us to hypothesize that these toxins may activate pro-inflammatory signalling cascades. Methodology and Principal Findings Here we demonstrate that palytoxin and a semi-purified Ostreopsis cf. ovata toxin extract obtained from a cultured strain isolated in the NW Adriatic Sea and containing a putative palytoxin and all the ovatoxins so far known – including the recently identified ovatoxin-f – significantly increase the levels of mRNAs encoding inflammation-related proteins in immune cells, i.e. monocyte-derived human macrophages, as assessed by Real-Time PCR analysis. Western immunoblot and electrophoretic mobility shift assays revealed that nuclear transcription factor -κB (NF-κB) is activated in cells exposed to toxins in coincidence with reduced levels of the inhibitory protein IκB-α. Moreover, Mitogen-Activated Protein Kinases (MAPK) were phosphorylated in response to palytoxin, as also reported by others, and to the Ostreopsis toxin extract, as shown here for the first time. By using specific chemical inhibitors, the involvement of NF-κB and p38 MAPK in the toxin-induced transcription and accumulation of Cycloxigenase-2, Tumor Necrosis Factor-α, and Interleukin-8 transcripts has been demonstrated. Conclusions and Significance The identification of specific molecular targets of palytoxin and its Ostreopsis analogues, besides contributing to expand the still limited knowledge of the intracellular signalling cascades affected by these toxins, may have important implications in setting up focused pharmacological interventions, replacing currently used symptomatic treatments.

Stereochemical Studies on Ovatoxin‐a

Ovatoxin-a is the main toxin produced by Ostreopsis ovata, a benthic dinoflagellate that has bloomed massively across the Mediterranean basin over the past years, inflicting both human and environmental suffering. Ovatoxin-a has recently been isolated from cultures of O. ovata and structurally identified as an analogue of palytoxin: in comparison with palytoxin, ovatoxin-a lacks three hydroxy groups at the 17-, 44- and 64-positions, but features an extra hydroxy functionality at the 42-position. Herein we report on the NMR-based elucidation of the stereochemistry of ovatoxin-a, which includes 7 stereogenic double bonds and 62 asymmetric carbon atoms. Understanding the full stereochemistry of ovatoxin-a is a step towards the elucidation of its mechanism of action on a molecular level.

Assignment of the absolute stereochemistry of oxazinin-1: application of the 9-AMA shift-correlation method for β-chiral primary alcohols

Abstract Reported herein is the assignment of the absolute stereochemistry of oxazinin-1, a cytotoxic compound recently isolated from the digestive glands of Mytilus galloprovincialis of the North Adriatic Sea. The recent 9-AMA shift-correlation method for assigning absolute configuration to β-chiral primary alcohols was employed and preparatory Molecular Mechanics calculations were carried out.

SxtA and sxtG Gene Expression and Toxin Production in the Mediterranean Alexandrium minutum (Dinophyceae)

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.