Chemical analysis of toxic microalgae from Mediterranean Sea

Marine plankton is constituted by microalgae existing either in unicellular forms or in colonies of cells. These invisible organisms play a key role in the aquatic ecosystem as they are the producers of organic material thus being the first ring of the aquatic food chain. Algal proliferation in the plankton, commonly referred to as algal bloom, is a periodic phenomenon occurring in many countries across the world usually in coincidence with particular climatic and environmental events. Blooms due to microalgae producing biotoxins are known as “harmful algal blooms”, as they pose serious threats to human health. In fact, oysters, mussels, clams and in general bivalve organisms, while feeding by filtering seawater, can accumulate toxins in their edible tissues sometimes to such a level to harass unaware consumers. Hence it can be stated that bivalves constitute a crucial ring of the food chain responsible for transferring toxicity from the plankton onto humans. During my PhD within Prof. Ciminiello’s research group, I investigated the toxic profile of the Mediterranean Sea where over the past decades a number of toxic microalgae have bloomed. In particular, I focused my attention on toxic outbreaks caused by microalgae belonging to the Ostreopsis genus, quite rife along the Italian coasts. Harmful algal blooms linked to Ostreopsis spp. reached alarming proportions in the late July of 2005 and 2006, when many people required extended hospitalization for respiratory distress after exposure to marine aerosol along the beach and promenade of Genoa. During these toxic episodes Prof. Ciminiello’s research group succeeded in identifying a putative palytoxin - the most potent non-proteic toxin so far known (DL50 <100 nanograms/Kg) - as well as a new palytoxin analog, ovatoxin-a, as the two main compounds responsible for the human poisoning. This was achieved by setting up a new analytical method for detecting palytoxin based on association of liquid chromatography with mass spectrometry (LC-MS). In the wake of this research on Ostreopsis spp. I carried out in-depth studies on O. ovata toxic profile succeeding in identifying four new palytoxin-like compounds, named ovatoxin-b, -c, -d, and, -e by High Resolution (HR)LC-MS and MS/MS experiments on a hybrid Linear Ion Trap (LTQ) Orbitrap XLTM FTMS operating up to 100,000 of resolution power. On the basis of the MS data in our hands we could also give some preliminary information about their structures; however NMR mono- and bi-dimensional experiments are needed to definitively assign their chemical architecture. Since over the last years the restating of the O. ovata summer blooms has raised serious concerns to both human health and economy, from October 2006 a commission appointed by the Ministero della Salute has been in charge of investigating and monitoring Ostreopsis phenomenon along the whole Italian coasts to the aim of evaluating risks to human health and preventing any possible human intoxication. So, from the summer of 2007 on, a program of monitoring in the frame of the project “Monitoraggio Ostreopsis ovata litorale costiero Regione Campania” was started by ARPA Campania in collaboration with Stazione Zoologica A. Dohrn, Istituto Zooprofilattico Sperimentale del Mezzogiorno, and with Dipartimento di Chimica delle Sostanze Naturali, where I carried out my PhD. Therefore, every summer from 2007 to 2010, during the O. ovata blooms I carried out LC-MS analyses on samples of both mussels and sea-urchins, collected along the Campania coasts, once proven positive to the mouse bioassay. For many Campania sites, for which our analyses showed toxin contents above the EFSA (European Food Safety Authority)-established tolerance limits (30 μg/Kg), mussel harvesting was banned. Such a ban was lifted only when our analyses highlighted toxin amounts in the investigated samples well below the above official limits. Considering the deep sanitary and economic impact due to the Ostreopsis outbreaks, I also tried to set up a suitable purification procedure for ovatoxin-a – which accounts for 50% of the total toxin content - with the purpose of obtaining sufficient amount of pure compound for investigating its toxicology as well as its mechanism of action. The best results were obtained by extracting ovatoxin-a from algal pellets with methanol and methanol/water 1:1, followed by partition of the water extract with chloroform, and then by a medium pressure chromatographic separation employing a reverse stationary phase and a UV instrument as detector, and eventually by a high pressure chromatographic separation using LTQ Orbitrap XL FTMS as detector. In this frame I also analyzed O. ovata cultures coming from the Adriatic (Ancona) and the Tyrrhenian Seas (Latina) in collaboration with the University of Bologna. Pellet samples and culture mediums were collected in both exponential and stationary cellular growth phases, extracted, and finally analyzed by LC-MS to the aim of evaluating the toxins production in the different Italian strains and at the different growth levels. Our studies showed that the Adriatic strain is richer in toxins than the Tyrrhenian one, and the toxins content during the stationary growth phase is higher than that of the exponential phase. In the field of this research on palytoxins, I also studied extracts from soft corals belonging to the Palythoa genus collected in Hawaii. In particular, I carried out LC-MS analyses on a triple quadrupole mass spectrometer at unit resolution, with addiction of NaCl and KCl to the mobile phase to induce adduct ions production. LC-MS experiments were also recorded on a time of flight instrument, operating at high resolution. The whole of the above studies allowed us to individuate a new palytoxin analogue whose molecular formula (C129H224N3O55) appeared to contain an oxygen atom more than palytoxin (C129H224N3O54). NMR mono- and bi-dimensional experiments on the new palytoxin analogue identified it as 42-OH palytoxin. Another subject of my research was the investigation of the toxin profile of Alexandrium. ostenfeldii, a dinoflagellate producing spirolides, fast acting toxins with an unusual 7-membered spiro-linked cyclic imine moiety. Beyond the fact that once injected intraperitoneally into a mouse spirolides give rise to neurologic symptoms, their human toxicity is largely unknown. A. ostenfeldii samples collected in the Adriatic Sea were extracted and analyzed by LC-MS/MS that highlighted the presence of some unreported spirolides on the basis of their characteristic fragmentation pattern. Finally, NMR studies allowed to characterize two of the new spirolides as 27-OH-13-desmetil spirolide C and 27-oxo-13,19-didesmetil spirolide C

Variability of toxin profile and content of Ostreopsis cf. ovata from the Mediterranean Sea

Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. In the last decade, O. confronta (cf.) ovata has become increasingly frequent with massive blooms with consequent relevant negative impacts on benthic communities and on human health through skin contact and toxic aerosols. Following the Mediterranean Ostreopsis-related outbreaks we developed a liquid chromatography-high resolution mass spectrometry (HRMS) method to be employed in the investigation of both field and cultured algal samples of O. cf. ovata. Our studies characterized O. cf. ovata as the producer of minute amounts of a putative palytoxin, one of the most potent marine toxins so far known, and of much higher amounts of several palytoxin congeners, that we named ovatoxins. Ovatoxin-a has been recently isolated and structurally elucidated, while ovatoxin-b, -c, -d, -e, and -f have not been isolated yet. Our recent research has been focused on characterization of toxin profiles of O. cf. ovata of different geographical origin that resulted to be quite different both qualitatively and quantivatively. We analyzed by LC-HRMS over 40 cultured strains of Ostreopsis cf. ovata collected along the Italian and the French Mediterranean coasts identifying basically 4 kinds of toxin profiles. In most of the strains ovatoxin-a dominates toxin profiles accounting for more than 50% of the total toxin content. But about 20% of the strains were found to produce only ovatoxin-a, d/e and putative palytoxin. Unique toxin profiles were found in a strain from Ancona (Marche, Italy) in which ovatoxin-f was the dominant toxin and a strain from Villefranche sur Mer (France) that did not produce ovatoxin-a, but just ovatoxin-b and c. These data trigger some questions on the reasons why toxin profiles and toxin content are so different as well as on toxins that need to be determined in monitoring programs of O. cf. ovata toxins in environmental and seafood sample

Liquid chromatography–high-resolution mass spectrometry for palytoxins in mussels

Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography–high resolution mass spectrometry (LC–HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14–1000 ng mL in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC–HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 μg kg in crude mussel extracts and 15 μg kg after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this

MICROALGHE BENTONICHE TOSSICHE:ISOLAMENTO, CARATTERIZZAZIONE STRUTTURALE E ANALISI QUALI-QUANTITATIVA DELLE TOSSINE PRODOTTE.

Questo progetto si configura come la naturale continuazione di una linea di ricerca già in corso presso i nostri laboratori sul monitoraggio e sull'identificazione della struttura chimica di biotossine algali presenti nelle acque costiere del territorio nazionale. L'individuazione e le determinazione strutturale delle tossine prodotte dalle alghe tossiche rappresenta un punto focale dell'indagine sui danni da esse arrecate all'uomo e all'ambiente e sulle misure da adottare per fronteggiare situazioni di emergenza legate alla loro fioritura. In particolare, il progetto di ricerca sarà focalizzato sullo studio della principale alga tossica che attualmente rappresenta il maggiore pericolo per la salute pubblica: l'alga tropicale Ostreopsis ovata. Negli ultimi anni, fioriture di questa specie algale sono state riportate con sempre maggiore frequenza lungo l'intero litorale marittimo italiano. In occasione delle fioriture sono stati riscontrati estesi danni alla fauna bentonica e disturbi respiratori in bagnanti o persone che stazionavano nei pressi dell'area interessata con sintomi come rinorrea, tosse, febbre, broncocostrizione con modeste difficoltà respiratorie, e in alcuni casi, congiuntivite. Molto probabilmente questi sintomi sono stati scatenati dall'inalazione di frammenti di O. ovata o di tossine prodotte da quest'alga presenti nell'aerosol marino. Sembra pertanto necessaria una approfondita indagine sui costituenti tossici prodotti da quest'alga tropicale, che al momento rappresenta una seria minaccia sia per l'ambiente marino, che per la salute pubblica. Inoltre, poiché in associazione con Ostreopsis possono proliferare altre specie che producono tossine diverse, quali Prorocentrum lima, che produce acido okadaico, tossina di tipo diarroico, e Coolia monotis, che produce tossine non ancora caratterizzate, le indagini saranno estese anche a queste microalghe. Studi preliminari LC/MS condotti dal nostro gruppo di ricerca analizzando sia campioni di plancton raccolti in coincidenza della proliferazione di O. ovata, che estratti di colture cellulari, hanno evidenziato che la principale tossina prodotta da tale microalga è l'ovatossina-a, una tossina strutturalmente simile alla palitossina. L'analisi LC/MS, tuttavia, anche se indica chiaramente la presenza di una sostanza palitossino-simile, non è in grado di definire i suoi dettagli strutturali e se l'alga produce una sola molecola o una miscela di analoghi. Analisi tese all'isolamento e alla caratterizzazione di tale composto, oltre che alla definizione completa del profilo tossico dell'alga, sono necessarie al fine di valutare attentamente l'impatto sulla salute del consumatore. La nostra indagine prevede, inizialmente, l'analisi di plancton e di colture di microalghe bentoniche, allo scopo di individuare eventuali biotossine marine e, nel caso queste siano presenti in quantità sufficienti, di isolarle in modo da poter fornire standard di composti puri utili alla ricerca farmacologica. I laboratori del gruppo di ricerca sono ben attrezzati a tale scopo e hanno già contribuito alla delucidazione strutturale di un certo numero di biotossine. Si presterà attenzione soprattutto a quelle biotossine che causano seri problemi sanitari, o perché disperse nell'aerosol, o perché contaminano i molluschi eduli. Le tossine marine saranno isolate e purificate da ceppi di organismi produttori coltivati dall'Unità operativa dell'Università di Ravenna. Le tossine purificate saranno utilizzate da altri gruppi afferenti al progetto per studi sulla tossicità e sul meccanismo d'azione. Lo studio sarà, inoltre, affiancato da un'attenta attività di monitoraggio della presenza di questa tossina in diverse matrici ambientali e biologiche (biomassa algale, acqua, aerosol, organismi acquatici), e da uno studio sul bioaccumulo delle tossine identificate in organismi acquatici eduli che vivono nelle aree interessate da fioriture di microalghe tossiche

High Resolution LC-MS of ovatoxins: a strategy to face an emerging threat to seafood consumers in the Mediterranean area

In the last decade, the benthic dinoflagellate Ostreopsis ovata has caused severe sanitary emergencies along the Mediterranean coasts of Europe. Liquid chromatography-mass spectrometry (LC-MS) played a key role in the identification of palytoxin as the parent toxin produced by O. ovata (Ciminiello et al., 2006). Palytoxin is one of the most potent non-protein marine toxins so far known (Deeds and Schwartz, 2010) as well as one of the most complex non-polymeric water-soluble molecules ever described (Ciminiello et al., 2011). Following this finding, a number of palytoxin analogues, that we named ovatoxins (a-f), have been found and identified basing on high resolution (HR) LC-MS evidence (Ciminiello et al., 2006, 2010). Among them, ovatoxin-a is the major component of O. ovata toxin profile, representing up to 55% of the total toxin content, with the remaining 46% being constituted by the whole of the other ovatoxins, and palytoxin being present in very minute amounts . Blooms of Ostreopsis spp. may result in ovatoxin contamination of seafood and, in order to prevent sanitary risks, the need exists to develop efficient extraction procedures to be coupled to rapid and sensitive monitoring methods of palytoxin-like compounds in seafood. In the present study, the best conditions for extraction of palytoxin-like compounds from seafood and their quantification by using liquid HR LC-MS will be presented

High Resolution LC-MS and MS/MS studies of Ostreopsis ovata toxin profile

In the last decade, the benthic dinoflagellate Ostreopsis ovata has caused severe sanitary emergencies along the Italian coasts. Liquid chromatography-mass spectrometry (LC-MS) played a key role in the identification of putative palytoxin as the parent toxin produced by O. ovata. Following this finding, a number of palytoxin analogues, named ovatoxin-a, -b, -c, -d, and -e, have been found and identified basing on high resolution (HR) LC-MS evidence. Among them, ovatoxin-a is the major component of O. ovata toxin profile, representing up to 55% of the total toxin content, with the remaining 46% being constituted by the whole of the other ovatoxins and putative palytoxin. HR LC-MS studies on O. ovata are continuing. Toxin profiles of different O. ovata strains appear sometimes different both qualitatively and quantitatively and the presence of further new ovatoxins is emerging. In addition, preliminary information on the structure of ovatoxins has been gained by an in-depth investigation of their HR LC-MS/MS spectra in comparison to that of palytoxin. A comparison between HR LC-MS/MS spectra of palytoxin and ovatoxin-a allowed us to individuate the regions and, in some cases, the sites where structural differences between these molecules occur. In view of the structural complexity of ovatoxins, the fragmentation pattern of palytoxin and ovatoxin-a could serve as useful template to characterize minor congeners that could be available in too small amounts to be studied by NMR

Identification of palytoxin-Ca2+ complex by NMR and molecular modeling techniques

More than 40 years after its isolation, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. The Na(+),K(+)-ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K(+) and remarkable rise of cytosolic Na(+) levels. In addition, a slight permeability to Ca(2+) is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca(2+) concentration gives rise to downstream events ultimately leading to cell death. The widely accepted recognition of the dependence of important cellular events on calcium ion concentration propelled us to investigate the occurrence of palytoxin-Ca(2+) complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca(2+) is preferentially coordinated. This study constitutes the first characterization of a calcium complex with palytoxin and, as such, is expected to support the investigation of the toxin molecular bioactivity

Ostreopsis ovata along the Campania coasts: determination of putative palytoxin and ovatoxin-a by LC-MS

Over the last few years, microalgae belonging to Ostreopsis genus have been blooming along Italian coastlines, causing serious concerns to both environment and public health. In fact, concurrently to the algal blooms, environmental sufferings involving mostly epibenthos were observed and symptoms of respiratory distress occured in people exposed to marine aerosols by recreational or working activities. Such a phenomenon was recorded for the first time during summer 1998 along the north-western Tuscanian coasts, but reached alarming proportions in late July 2005, when about 200 people exposed to marine aerosols on the beach and promenade of Genoa required extended hospitalization. Human illness was concurrent with a massive bloom of Ostreopsis ovata along the Genoa coasts and disappeared as O. ovata population decreased. From 2006 onwards, O. ovata outbreaks have been observed every single year along the whole Tyrrhenian coasts from Liguria to Sicily as well as in the Adriatic Sea. These episodes caused great concern as Ostreopsis species are reported to produce palytoxin-like compounds. Palytoxin is a complex polyhydroxylated water-soluble compound and one of the most potent non-protein marine toxins so far known. A method for determination of palytoxin, based on combination of liquid chromatography and mass spectrometry (LC-MS), was developed by our group and used to investigate toxin profile of plankton collected along the Genoa coasts during 2005 and 2006 toxic outbreaks. In all O. ovata samples, the presence of two main toxins was highlighted: putative palytoxin and a much more abundant palytoxin-like compound never reported so far, ovatoxin-a; this latter shares part structure A with palytoxin. In 2007, Regione Campania has started a monitoring program to investigate the presence of O. ovata along the Campania coastlines. In this frame, we set up extraction methods for fast and accurate analyses of both seawater and seafood samples. A relevant number of plankton, mussels and sea-urchins samples collected at various sites were analyzed by LC-MS and the presence of putative palytoxin and ovatoxin-a was ascertained, indicating that both toxins had entered the marine food web with potential risk for the health of seafood consumer

Effects of N and P availability on carbon allocation in the toxic dinoflagellate Ostreopsis cf. ovata

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata are usually associated with shallow and calm coastal waters, characterized by low nutrient concentrations. The algal cells typically cover the benthic substrates, such as the macroalgal and invertebrate communities and rocks, forming a mucilaginous film. Data reported on O. cf. ovata toxin production observed under both field and culture conditions show high variability in terms of toxic profile and cellular content; little is known about the environmental and physiological aspects which regulate the toxin dynamics. In this study, O. cf. ovata physiology was investigated using batch cultures supplied with nutrient concentrations similar to those found in the Adriatic Sea during the recurrent blooms and the observed cellular dynamics were compared with those found in a culture grown under optimal conditions, used as a reference. Data on the cellular C, N and P content during the growth highlighted a possible important role of the cellular nutritional status in regulating the toxin production that resulted to be promoted under specific intervals of the C:N and C:P ratios. The variable toxicity found for O. cf. ovata in various geographic areas could be related to the different in situ prevalent environmental conditions (e.g., nutrient concentrations) which affect the cellular elemental composition and carbon allocation. The obtained results strongly suggest that in the environment toxin production is steadily sustained by a low and constant nutrient supply, able to maintain appropriate cellular C:N (>12) or C:P (>170) ratios for a long period. These results explain to some extent the variability in toxicity and growth dynamics observed in blooms occurring in the different coastal areas