Dissertations / Theses on the topic 'Palytoxins'

2010/2011
The present thesis was developed for the characterization of the risk associated to palytoxins as seafood contaminants. To this aim, an integrated approach between in vitro and in vivo studies was chosen. Palytoxin and its analogues are known seafood contaminants that can accumulate in several edible species of shellfish, fish, crustaceans and echinoderms. Generally, primary symptoms associated to the ingestion of contaminated food involve the gastro-intestinal apparatus and later develop with the involvement of the muscular tissue. For a better comprehension of the mechanism of action of this family of biotoxins, the effects of PLTX have been studied on primary culture of mouse skeletal muscle cells. The myotoxic insult triggered by PLTX was described in detail with the definition of the cytotoxicity together with the description of the morphologic alterations and functional impairment caused by the toxin. Moreover, the influence of the ionic composition of the extracellular medium on the effects of the toxin was elucidated. Primary cultures of skeletal muscle cells, that presents in vitro many of the peculiarities of the adult muscle fiber, allowed the investigation of the membrane mechanisms that regulate the intracellular calcium increase triggered by the toxin. It was possible to discriminate the difference between calcium release from intracellular stores and the calcium entrance from extracellular compartment both elicited by the toxin, and to understand the importance of the latter in relation to the toxic event. Moreover, the involvement of the main membrane channels and transporters that may be related to the entrance of calcium was investigated and the crucial role of stretch-activated channels in the mechanism of toxicity was demonstrated. Once defined the crucial molecular mechanisms of action of PLTX, experiments were also performed with two of its analogue: the 42-hydroxyl-palytoxin and the ostreocin-D. In parallel to in vitro studies, the effects of repeated oral administration of PLTX in mice were also investigated. In fact, in vitro studies are not sufficient for the complete comprehension of the real hazard associated to a food contaminant, since molecules once in contact with the body may undergo adsorption, distribution and metabolism before reaching the target tissue. Short-term (7 days) administration of the toxin revealed toxicity at all the doses tested and lethality was recorded in the treated animals already from the dose of 30 µg/kg. Histological analysis highlighted alterations in several tissues: severe inflammatory processes and even foci of necrosis were observed in lungs. Alteration of the muscular tissues was visible as fiber separation and degeneration in the heart and increased cellularity between fibers in skeletal muscle. Moreover, depletion of glycogen content of hepatocytes and moderate alterations of the spleen were also observed. Data collected in the present project revealed, for the first time, toxicity of PLTX at doses much lower than that currently used by European Food Safety Authority (EFSA) for the estimation of limit values for presence of these compounds in seafood. For this reason, these results are likely to have a considerable impact at regulatory level and to have crucial importance for the protection of the consumers.
Il presente lavoro di tesi è stato sviluppato con lo scopo di caratterizzare il rischio associato alla presenza delle palitossine quali contaminanti dei prodotti ittici destinati ad uso alimentare. A tal fine, è stato scelto un approccio integrato tra studi in vitro e in vivo. La palitossina e i suoi analoghi sono noti contaminanti dei prodotti ittici e possono accumularsi in diverse specie edibili di molluschi, pesci, crostacei ed echinodermi. Generalmente, i primi sintomi associati all’ingestione di cibo contaminato coinvolgono l’apparato gastro-intestinale e poi si sviluppano con l’interessamento del tessuto muscolare. Per una migliore comprensione del meccanismo d’azione di questa famiglia di biotossine, gli effetti della PLTX sono stati studiati mediante colture primarie murine di cellule muscolari scheletriche. L’insulto miotossico indotto dalla PLTX è stato descritto nel dettaglio con la definizione della citotossicità assieme alla descrizione delle modifiche morfologiche e delle alterazioni funzionali causate dalla tossina. Inoltre, è stata caratterizzata l’influenza della composizione ionica dell’ambiente extracellulare negli effetti della tossina. Le colture primarie di cellule muscolari scheletriche, che presentano molte delle caratteristiche peculiari della fibra muscolare adulta, hanno permesso l’indagine dei meccanismi che regolano l’aumento intracellulare di calcio indotto dalla tossina. E’ stato possibile discriminare la differenza tra il rilascio di calcio dai depositi intracellulari e l’entrata di calcio dai compartimenti extracellulari, entrambi effetti indotti dalla tossina, e comprendere l’importanza del secondo meccanismo in relazione agli eventi tossici. E’ stato indagato il coinvolgimento dei canali e trasportatori di membrana in relazione all’ingresso di calcio ed è stato dimostrato il ruolo cruciale dei canali attivati da stiramento nel meccanismo di tossicità. Una volta definiti i meccanismi d’azione cruciali per la PLTX, esperimenti sono stati condotti anche con due dei suoi analoghi, la 42-idrossi-palitossina e l’ostreocina-D. Parallelamente agli studi in vitro, sono stati studiati gli effetti della somministrazione orale ripetuta della PLTX nel topo. Infatti, gli studi in vitro non sono sufficienti alla comprensione del reale pericolo associato ad un contaminate alimentare, dal momento che, le molecole, una volta a contatto con l’organismo, possono subire assorbimento, distribuzione e metabolismo prima di raggiungere il tessuto bersaglio. La somministrazione a breve termine della tossina (7 giorni) ha rivelato tossicità a tutte le dosi somministrate, e letalità è stata registrata negli animali trattati già alla dose di 30 µg/kg. Le analisi istologiche hanno evidenziato alterazioni a carico di diversi tessuti: a livello polmonare sono stati osservati severi processi infiammatori associati anche a focolai di necrosi. Le alterazioni del tessuto muscolare erano visibili come degenerazione e separazione delle fibre nel cuore e aumento degli elementi cellulari tra le fibre del muscolo scheletrico. Inoltre sono stai osservati, deplezione del contenuto di glicogeno negli epatociti e moderate alterazioni della milza. I dati raccolti nel presente elaborato hanno rivelato, per la prima volta, la tossicità della palitossina a dosi molto inferiori rispetto a quelle correntemente utilizzate della European Food Safety Authority (EFSA) per la stima di valori limite per la presenza di questi composti nei prodotti ittici destinati ad uso alimentare. Per questo motivo, i risultati avranno probabilmente un considerevole impatto a livello legislativo e un’importanza cruciale per la protezione dei consumatori.
XXIV Ciclo
1984

Cette thèse confidentielle public/privé porte sur l’étude des sources biologiques de la palytoxine (PlTX) dont l’identification est nécessaire afin de développer un protocole industriel standardisé permettant d’obtenir des quantités importantes de toxine pour réaliser diverses études. Premièrement, l’activité anticancéreuse in vitro de la PlTX a été évaluée sur des lignées cellulaires cancéreuses humaines et murines. La cytotoxicité de la PlTX est 106 fois plus importante sur les lignées cancéreuses par rapport aux lignées non cancéreuses témoins. La PlTX utilisée a été purifiée à partir d’une espèce encore non décrite de Palythoa, qui a été caractérisée phylogénétiquement et morphologiquement et qui fournit les quantités de PlTX pures les plus élevées à ce jour (2,8 ± 0,45 mg/g de matière fraîche). La localisation de la PlTX dans les tissus de cette espèce a été décrite par des techniques innovantes d’imagerie par spectrométrie de masse MALDI. Deuxièmement, une étude phylogénétique et une quantification de la PlTX par HPLC chez 29 échantillons de Zoantharia a été réalisée. La spectrométrie de masse ESI a permis de donner la structure chimique du type de PlTX purifiée à partir de ces échantillons. La dernière partie présente une étude préliminaire sur la culture ex hospite des endosymbiotes unicellulaires de Palythoa spp. appartenant au genre Symbiodinium afin de tester leur implication dans la synthèse de PlTX. En conclusion, il ressort que la PlTX présente un effet anticancéreux très prometteur. Le moyen le plus efficace actuellement pour obtenir de la PlTX en quantité importante est de l’extraire à partir d’une espèce de Palythoa caractérisée grâce à ce travail
The work presented in this manuscript is the outcome of a confidential public/private collaboration. It concerns the study of biological sources of PlTX. Their identification is required to develop an industrial-standard protocol in order to obtain sufficient quantity of toxin to perform several studies. In the first part, the in vitro anti-cancer activity of PlTX was evaluated upon several human and murine cancerous cell lines. The cytotoxic activity of PlTX was 106-times more powerful against cancerous lines compared to non-cancerous control lines. The PlTX used in this work was purified from an undescribed species of Palythoa which was characterized based on phylogenetic and morphological analyses. This species produces the highest PlTX levels ever recorded (2.8 ± 0.45 mg/g fresh matter). PlTX localization in tissues of this Palythoa species was also described by innovative MALDI mass spectrometry imaging techniques. The second part presents a phylogenetic study of 29 samples of Zoantharia and quantification of PlTX by HPLC. The ESI-mass spectrometry allowed to characterize the chemical structure of the type of PlTX purified from these samples. The last part of this thesis constitutes a preliminary study on the ex hospite culture of unicellular endosymbionts belonging to the genus Symbiodinium. Ensosymbiont cells were isolated from Palythoa species containing high PlTX concentrations in order to test their implication in PlTX synthesis. In conclusion, PLTX has a very promising anticancer activity, and the best way to obtain large quantities of PlTX is the extraction from a Palythoa species characterized in this work

2010/2011
Palytoxin (PLTX) is a marine toxin identified in Palythoa zoanthid corals and Ostreopsis dinoflagellates, representing an increasing hazard for human health. Human poisonings attributed to PLTX exposure are usually associated to ingestion of contaminated seafood and to marine aerosol exposure during Ostreopsis blooms. However, also dermatological problems have been recently associated to PLTX cutaneous exposure during Ostreopsis blooms as well as after handling of Palythoa corals. Despite the increasing human cases of dermotoxicity attributed to PLTX, very few data about its dermal toxicity are presently available. Hence, the aim of this study is to investigate the cutaneous effects of PLTX characterizing its mechanism of action. Thus, this toxicological in vitro study has been carried out on spontaneously immortalized human keratinocytes (HaCaT cells), as a first-round screening of dermotoxicity. The entity of cytotoxicity induced by PLTX has been firstly investigated. A short time exposure (4 h) to PLTX reduces mitochondrial activity (MTT assay), cell mass (SRB assay) and plasma membrane integrity (LDH leakage) with different potencies (EC50 values of 6.1±1.3x10-11, 4.7±0.9x10-10 M and 1.8±0.1x10-8 M, respectively). All these effects are ouabain-sensitive corroborating the dependency of PLTX effects on the interaction with Na+/K+-ATPase. These results indicate that among the chain of intracellular events following the interaction of PLTX with the Na+/K+-ATPase the earliest is mitochondrial damage. This sustained cytotoxic effect can be explained by the high affinity of binding to HaCaT cells. Indeed, saturation experiment reveals a Kd affinity constant of 3.0±0.4x10-10 M after an exposure time as short as 10 minutes. A possible mechanism of mitochondrial dysfunction can be reactive oxygen species (ROS) overproduction. Among all, only superoxide anion (O2-) seems to be produced by the toxin after only 1 h, whereas neither nitric oxide nor peroxynitrite formation are detected. Hence, the mechanism of O2- production has been investigated. Real time PCR analysis together with western blot analysis suggest a possible involvement of NADPH oxidase (NOX) and inducible nitric oxide synthetase (iNOS) since an early increase of their gene and protein expression was observed after short (1 – 4 h) but not longer (24 h) exposure times. On the contrary, other enzymes involved in ROS production (i.e. COX-1, COX-2, XOD) seem to be not involved in PLTX effects. Moreover, using selective inhibitors of these enzymes, we found that only DPI, a nonspecific inhibitor of both NOX and NOS, is able to inhibit by 15%, 26% and 43% O2- production induced by 10-10, 10-9 and 10-8 M PLTX, respectively. However, NMMA, inhibitor of NOS, significantly reduces only O2- produced by high (10-8 M) but no by low (10-9 and 10-10 M) PLTX concentrations, whereas the selective inhibitor of NOX apocynin is totally ineffective. Moreover, since their co-administration does not reproduce DPI effect, a prominent role of these enzymes in causing PLTX-induced oxidative stress seems unlikely. Another feasible source of O2- is mitochondria itself and its production is regulated by H+ fluxes through mitochondrial membranes. Indeed, in presence of nigericin, an ionophore that reduces the H+ imbalance, PLTX-induced O2- is significantly reduced by 23% (10-9 M PLTX) and 24% (10-8 M PLTX). Furthermore, the co-administration with rotenone, a complex I inhibitor, that per se is ineffective, results in a further inhibition of O2- production (-32% and -43% in the presence of 10-9 and 10-8 M PLTX, respectively). Moreover, O2- production turned out to be ouabain-sensitive and Na+-dependent but Ca2+-independent. Thus, on the basis of these results it has been hypothesized that PLTX binding to Na+/K+-ATPase induces intracellular overload of Na+ followed by intracellular increase of H+ with a consequent ΔpH increase across H+-impermeable mitochondrial inner membrane and O2- overproduction by reverse electron transports through mitochondrial chain. Under oxidative stress conditions, mitochondrial dysfunction can be mediated by mitochondrial permeability transition pore (MPTP), which opening, indeed, is induced by PLTX already after only 5 minutes exposure. MPTP opening, which turned out to be cyclosporine A-independent, seems to be mainly induced by the sustained ionic imbalance, since in Na+-free, Ca2+-free medium and in presence of nigericin PLTX effect is strongly inhibited. The very rapid Na+-dependent opening of MPTP suggests that this is the peculiar mechanism of PLTX cytotoxicity and cell death primum movens. Cell death induced by the toxin seems to occur with necrotic-like features. PLTX, indeed, induces a concentration- and time-dependent as well as irreversible uptake of PI after only 1 h exposure and confocal images revealed dramatic morphological alterations such as plasma membrane ruptures and leakage of cytolpasmic content after 4 h. By contrast, caspasis 3/7, 8 and 9 are not activated by PLTX up to 24 h, neither under recovery conditions. Moreover, apoptotic bodies formation is not observed, discarding apoptosis occurrence. Finally, PLTX effects on some pro-inflammatory mediators such as cytokines (IL-1α, IL-6, IL-8 and TNF-α) and arachidonic acid metabolism products (PGE2 and LTB4) have been evaluated. The toxin (10-11 M) induces an early release of PGE2 that is time-dependent after 2 h exposure. On the contrary, even if an early gene expression (1–4 h) is observed, the toxin induces a delayed release of IL-6 and IL-8 (24 h), whereas no effects have been observed evaluating IL-1α and TNF-α. In conclusion, this study highlights the toxic in vitro properties of PLTX on human keratinocytes. The intracellular pathway of the sustained PLTX cytotoxicity leading to cell death has been characterized, as well as the inflammatory mediators involved in skin irritant properties of the toxin. These results can corroborate the use of non steroidal anti-inflammatory drugs in association with anti-inflammatory corticosteroids.
La palitossina (PLTX) è una tossina marina identificata in coralli zoantidi appartenenti al genere Palythoa e dinoflagellati del genere Ostreopsis. Intossicazioni umane attribuite alla PLTX sono state solitamente associate all'ingestione di prodotti ittici contaminati, nonché da un'esposizione ad aerosol marino durante le fioriture di Ostreopsis. Tuttavia, anche problemi dermatologici sono stati recentemente associati alla PLTX in seguito ad esposizione cutanea durante fioriture di Ostreopsis o manipolando coralli Palythoa. Nonostante i crescenti casi di dermotossicità attribuiti alla PLTX, pochissimi dati sulla sua tossicità cutanea sono attualmente disponibili. Lo scopo di questo studio è stato, pertanto, indagare gli effetti cutanei della PLTX caratterizzando il suo meccanismo d'azione. E’ stato quindi effettuato uno studio tossicologico in vitro su cheratinociti umani spontaneamente immortalizzati (cellule HaCaT), considerate metodo predittivo per uno screening preliminare di dermotossicità. In primo luogo è stato caratterizzato il grado di citotossicità indotta dalla tossina. Un breve tempo d'esposizione (4 h) alla PLTX riduce l'attività mitocondriale (saggio MTT), la massa cellulare (saggio SRB) e l'integrità della membrana plasmatica (perdita LDH) con diversi valori di EC50 (6.1 ± 1.3x10-11, 4.7 ± 0.9x10-10 M e 1.8 ± 0.1x10-8 M, rispettivamente). Tutti questi effetti sono sensibili alla ouabaina, corroborando la dipendenza degli effetti della PLTX sull'interazione con la Na+/K+-ATPasi. Questi risultati indicano che fra la catena di eventi intracellulari dopo l'interazione con l’ATPasi il più sensibile è un danno mitocondriale. Questo effetto può essere spiegato dall’alta affinità di legame della tossina con le cellule HaCaT. Infatti, esperimenti di saturazione rivelano una costante di affinità (Kd) pari a 3,0 ± 0.4x10-10 M dopo un tempo di esposizione molto breve (10 minuti). Uno dei possibili meccanismi di disfunzione mitocondriale è una sovrapproduzione di specie reattive dell'ossigeno (ROS). Tra tutti, solo l’anione superossido (O2-) sembra essere prodotto dalla tossina dopo 1 h, mentre né ossido nitrico né formazione di perossinitrito sono stati rilevati. Quindi, il meccanismo di produzione di O2- è stato studiato. Analisi real time-PCR ed analisi western blot suggeriscono un possibile coinvolgimento della NADPH ossidasi (NOX) e della forma inducibile dell’ossido nitrico sintetasi (iNOS) poiché un aumento precoce della loro espressione genica e stata osservata dopo brevi (1 - 4 h) ma non lunghi (24 h) tempi di esposizione. Al contrario, altri enzimi coinvolti nella produzione di ROS (COX-1, COX-2, XOD) sembrano non essere coinvolti nel meccanismo di produzione di O2- da parte della tossina. Inoltre, tramite l'utilizzo di inibitori selettivi di questi enzimi, è emerso che solo il DPI, un inibitore non specifico sia di NOX che di NOS, è in grado di inibire del 15%, 26% e 43% la produzione di O2- indotta da 10-10, 10-9 e 10-8 M PLTX, rispettivamente. Tuttavia, l’NMMA, inibitore delle NOS, riduce in modo significativo solo O2- prodotto da alte (10-8 M), ma non basse (10-9 e 10-10 M) concentrazioni di PLTX, mentre l'inibitore selettivo delle NOX apocinina è totalmente inefficace. Inoltre, poiché la loro co-somministrazione non riproduce l’effetto inibitorio del DPI, un ruolo preminente di questi enzimi nel causare stress ossidativo sembra improbabile. Un'altra fonte possibile di O2- è il mitocondrio. La sua produzione è regolata dal flusso di H+ attraverso le membrane mitocondriali. Infatti, in presenza di nigericina, uno ionoforo che riduce lo squilibrio protonico, i livelli di O2- indotti dalla PLTX vengono significativamente ridotti del 23% (10-9 M PLTX) e 24% (10-8 M PLTX). Inoltre, la co-somministrazione con il rotenone, un inibitore del complesso I della catena mitocondriale di trasporto degli elettroni, che è di per sé inefficace, induce un’ulteriore inibizione di produzione di O2- (-32% e -43% in presenza di 10-9 e 10-8 M PLTX, rispettivamente). Inoltre, la produzione di O2- risulta essere ouabaina-sensibile e Na+-dipendente, ma Ca2+-indipendente. Pertanto, sulla base di questi risultati è stato ipotizzato che il legame della PLTX con la Na+/K+-ATPasi induce un aumento intracellulare di Na+ seguito da aumento intracellulare di H+ con un conseguente aumento di ΔpH attraverso la membrana mitocondriale interna con una sovrapproduzione di O2- indotta dal trasporto inverso degli elettroni attraverso la catena mitocondriale. In condizioni di stress ossidativo, la disfunzione mitocondriale può essere mediata dall’apertura dei pori di transizione mitocondriali (MPTP). La loro apertura, infatti, viene indotta dalla PLTX già dopo soli 5 minuti di esposizione. Tale apertura, che si è rivelata ciclosporinaA-indipendente, sembra principalmente indotta dallo squilibrio ionico indotto dalla tossina, poiché in terreni privo di Na+ e privo di Ca2+ e in terreno contenente nigericina, l’attività della tossina è fortemente inibita. La rapidissima apertura di MPTP suggerisce che questo è il peculiare meccanismo di citotossicità della tossina e il primum movens della cellule morte. La morte cellulare sembra verificarsi con un danno necrotico. La PLTX, infatti, induce un uptake di PI (marker di necrosi) in maniera concentrazione e tempo-dipendente. Tale uptake è inoltre irreversibile, dopo solo 1 h di esposizione e immagini ottenute al microscopio confocale rivelano drammatiche alterazioni morfologiche, quali rotture della membrana plasmatica e la perdita di contenuto citoplasmatico dopo 4 h. Al contrario, le caspasi 3/7, 8 e 9 non sono attivate dalla PLTX fino a 24 h, né sotto condizioni di recovery. Inoltre, la formazione di corpi apoptotici non è stata rilevata, scartando l’ipotesi di una morte di tipo apoptotico. Infine, gli effetti della PLTX su alcuni mediatori proinfiammatori quali citochine (IL-1α, IL-6, IL-8 e TNF-α) e metaboliti dell’acido arachidonico (PGE2 e LTB4) sono stati valutati. La tossina (10-11 M) induce una rapida produzione di PGE2 che è tempo-dipendente dopo 2 ore di esposizione. Al contrario, la tossina induce un rilascio ritardato di IL-6 e IL-8 (24 h), anche se alterazioni dell'espressione genica si sono osservate dopo breve tempo di contatto con la tossina (1-4 h). mentre non sono stati osservati effetti valutando IL-1α e TNF -α. In conclusione, questo studio mette in evidenza le proprietà tossiche in vitro della PLTX su cheratinociti umani. L’elevata citotossicità indotta dalla tossina conduce ad una morte cellulare di tipo necrotico mediata dai mitocondri. Infine, i mediatori infiammatori coinvolti nella proprietà irritanti della pelle della tossina sono stati caratterizzati, ponendo delle basi molecolari per spiegare l'utilizzo di farmaci anti-infiammatori non steroidei in associazione con corticosteroidi.
XXIV Ciclo
1983

Palytoxin, the most potent known marine toxin, has been demonstrated to produce acute neurological disturbances in several animal species and, possibly, even in humans. However, the effects of palytoxin on excitable membranes have not been well characterised or explained. Palytoxin occurs within the same ecosystem in which other seafood toxins such as ciguatoxin, saxitoxin and brevetoxins are found. Also, several reports suggest that, occasionally, palytoxin may contribute to the ciguatera syndrome. As these other toxins are known to affect the sodium ion channel, either by activation or inactivation, evidence that palytoxin may also affect this structure was sought. The studies performed on the ventral coccygeal nerve of the rat tail, subsequent to an intraperitoneal injection of palytoxin, demonstrated a significant slowing of mixed nerve and motor conduction velocities and a reduction in the amplitude of the mixed nerve and motor action potentials. However, palytoxin exerted no effect on the latency of the shortest F-wave response. In the palytoxin-treated group, the absolute and relative refractory periods were prolonged. Palytoxin also appeared to induce a significant prolongation of the supernormal period of nerve excitability. The response of the palytoxin-treated ventral coccygeal nerve to repetitive stimulation demonstrated no consistent abnormality. These studies lend indirect support to the proposition that at least one action of palytoxin is that of an alteration in the excitability of neural tissue by inducing persistent sodium ion channel activation. Lignocaine, administered via the intraperitoneal route, was demonstrated to reverse many of the electrophysiological disturbances and, particularly, the prolonged supernormal period, in palytoxin-treated rats. These results suggest that prior activation of sodium ion channels by palytoxin may be blocked by lignocaine. Ethanol, administered via the intraperitoneal route, was observed to reverse many of the electrophysiological disturbances recorded in the palytoxin-treated rats, however, the supernormal period remained prolonged, yet not particularly enhanced or diminished in magnitude. Ouabain, administered via the intraperitoneal route, also reversed many of the electrophysiological disturbances induced in palytoxin-treated rats. However, in these animals, the palytoxin-induced supernormal period remained prolonged, yet not exaggerated or diminished in magnitude. These results suggest that membrane excitability may occur independently of, or with, the (Na+,K+)A TPase mechanism. The effect of palytoxin on mammalian nerve tissue appeared to be modified at a lower temperature. Mixed nerve and motor conduction, F-wave responses, absolute and relative refractory period and supernormal period studies were performed on palytoxin-treated animals at 25°C. The only significant abnormalities were a prolongation of the relative refractory period and of the supernormal period. This study suggests that, at least, in mammalian nerve tissue, this toxin is less active at a lower ambient temperature. In terms of central studies, in palytoxin-treated rats the brainstem auditory evoked response was not significantly altered although there was significant prolongation of the corticospinal evoked response. This finding suggests that palytoxin is capable of crossing the blood-brain barrier and, then, exerting an effect on central nerve membranes. These results are similar to those induced by ciguatoxin in the ventral coccygeal nerve of the rat. Both toxins produce slowing of the mixed nerve and motor conduction velocities, a reduction in the amplitude of the mixed nerve and motor action potentials and a prolongation of the refractory period and supernormal period. Lignocaine abolishes the supernormal period induced by both toxins. At a temperature of 25°C, both toxins produce reduced electrophysiological effects, in comparison with those produced at 37°C. Ciguatoxin is known to bind to and to activate or open sodium ion channels. These studies suggest that at least one action of palytoxin may be that of a similar activation of sodium ion channels. With reference to these studies and to the fact that both toxins occur within the same ecosystem, palytoxin could potentially contribute, on occasions, to the ciguatera syndrome.

2012/2013
The increasing distribution of marine microalgae which may produce toxins poses concern on their possible accumulation in seafood, with possible toxic effects in humans after its consumption. Also in the Mediterranean Sea, microalgae producing okadaic acid (OA) and its analogues, the main diarrheic toxins contaminating edible shellfish, were frequently detected since several years. In addition, blooms of potentially toxic microalgae belonging to Ostreopsis genus, producing palytoxins (PLTXs), were also reported in the recent years. Simultaneously, PLTX and its analogue ovatoxin-a were identified in microalgal, shellfish and echinoderm samples. This phenomenon could represent a risk for human health since food borne intoxications, including some lethal cases, attributed to the consumption of seafood contaminated by palytoxin-like compounds, were reported in tropical areas. Furthermore, co-presence of microalgae producing OA and PLTXs and the possible consequent seafood contamination lead to consider the problem of toxic effects in humans due to a simultaneous exposure to both the toxins, which could induce synergistic effects. Thus, the aim of this study was to investigate the acute and short-term oral toxicity in mice by co-exposure to palytoxin and okadaic acid, in comparison to that of each single toxin. In particular, its aim is to identify not only the main toxic effects and the target organs, but also a NOAEL (No Observed Adverse Effect Level), useful in the assessment of subchronic toxicological risk, a situation that most likely corresponds to the toxin exposure by humans. The studies were carried out using female CD-1 mice (18-20 g body weight, 4 weeks old; Harlan Laboratories; S. Pietro al Natisone, Udine, Italy). All experiments were carried out at the University of Trieste, Italy in compliance with the Italian Decree n. 116/1992 as well as the EU Directive 2010/63/EU and the European Convention ETS 123. Initially, the effects of the acute oral administration of palytoxin (30, 90 and 270 g/kg) combined to OA (370 g/kg) were studied in comparison to those of the same doses of the single toxins. After gavage administration of the toxin or vehicle (phosphate buffered saline, containing 1.8 % ethanol; controls) to groups of 8 mice, the animals were monitored for 24 h (5 mice/dose) or 14 days (3 mice/dose) for symptoms. Aftr death or sacrifice, they were submitted to necropsy, taking also blood samples for hematochemical analysis and the main organs and tissues for the histological analysis by light microscopy. Within 24 h from the administration, toxic effects and lethality were recorded only in mice administered with the higher doses of PLTX (90 or 270 g/kg), alone or in combination with OA (370 g/kg). In addition, lethality was recorded only at the highest PLTX dose alone or combined with OA. The signs and symptoms recorded in mice (scratching, piloerection, abdominal swelling, ataxia, paralysis of the hind limbs, dyspnoea) were slightly more pronounced in mice administered with both the toxins with respect to those recorded in mice administered with PLTX alone. At 24 h from PLTX administration (90 or 270 g/kg), alone or with OA, necropsy showed redness and fluid accumulation in the small intestine and an increased liver weight, whereas histological analysis showed changes at the forestomach (slight ulcers and inflammation) and liver (reduced glycogen content). During the whole observation period, mice treated with the higher doses of PLTX, also combined with OA, showed a reduced body weight and food consumption, while no toxic effects were recorded after 14 days from the administration. Thus, the study showed a slight additive effect between PLTX and OA after acute oral administration, estimating a NOAEL of 30 g/kg for PLTX and 370 g/kg for OA in combination or as single toxins. Due to the lack of toxicity data on PLTX after repeated oral administration, a toxicity study on the toxin alone after its daily administration for 7 days was carried out. The toxin was administered to groups of 6 or 8 mice at four doses (3, 30, 90 or 180 g/kg/day) and the animals were observed up to 24 h after the last treatment or, for subgroups of 3 mice, up to 14 days. The toxin induced lethal and/or toxic effects at the dose of 30 g/kg/day and above, starting from the third day of treatment; some mice died also during the recovery period after the toxin administration. At these doses, a significant reduction of body weight, abdominal swelling, chromodacryorrhea, piloerection, dyspnoea, sedation and/or paralysis of the hind limbs were recorded during the treatment period. Necropsy revealed gastrointestinal changes (gastric ulcers and intestinal fluid) in mice died during the treatment period, while histological analysis showed lung inflammation, locally associated with necrosis, hypereosinophilia and separation of myocardial fibers and liver changes (reduced glycogen content and necrosis at the highest dose). These data allowed an estimation of a provisional NOAEL corresponding to 3 g/kg/day, with the evidence of a steep dose-response relationship. The last step of the research included the study of the toxic effects in mice induced by 7days oral administration of PLTX (3 and 10 g/kg/day) and OA (185 g/kg/day) association. Both the doses of PLTX combined to OA induced lethal effects, with signs or symptoms including scratching, dyspnoea, paralysis of the hind limbs and body weight loss. At 24 h from the last treatment, necropsy showed also the presence of fluid in the small intestine of mice administered with the highest dose of PLTX combined to OA, accompanied with a decreased liver weight. An effect on the liver was also evidenced by the increased transaminases serum levels and, in one mouse, by slight foci of necrosis, associated to thymus atrophy. On the contrary, no toxic effects were recorded after 14 days from the treatment, with the exception of a decreased body weight in mice administered with both PLTX doses combined to OA. Therefore, the study showed a slight potentiation of the toxic effects by the repeated oral co-exposure to PLTX and OA, which induced lethal and/or toxic effects that were not recorded after the administration of the single toxins. The overall results highlighted that the concomitant presence of palytoxin and okadaic acid, even if chemically different, could increase their toxicity profile and latent effects. Although no clearly evident synergic or additive effects were observed, they cannot be ruled out. The next steps would include a confirmatory study in a larger group of animals followed by a 14- and 28 day repeat dose study. Additional studies which may be useful in evaluating ‘true’ and realistic conditions of intoxications which should also include animal studies aimed to investigate the impact of age and of impairment of the gastrointestinal tract on the toxicity of seafood toxins. In addition, they should consider that the amount of contaminated seafood reported in the existing guidelines does not always represent the true amount ingested by the consumers, underlining the importance of assessing the exposure to low doses of toxins as done in these studies.
La crescente frequenza di proliferazioni di microalghe marine in grado di produrre tossine, pone il problema dell’accumulo di tali composti nei prodotti ittici, con possibili effetti tossici per l’uomo in seguito al loro consumo. Anche nel mare Mediterraneo, da anni viene rilevata la presenza di microalghe produttrici di acido okadaico (AO) e suoi analoghi, tossine diarroiche che possono contaminare i molluschi destinati all’alimentazione. Recentemente, si sono anche verificate anche proliferazioni di microalghe appartenenti al genere Ostreopsis, produttrici di palitossine). Contemporaneamente, nei campioni di microalghe, di molluschi ed echinodermi sono state rilevate la palitossina (PLTX) ed un suo analogo, l’ovatossina-a. Ciò pone il problema del rischio per l’uomo, poichè in aree tropicali sono state riportate intossicazioni alimentari, anche letali, attribuite all’assunzione di prodotti ittici contaminati da composti palitossino-simili. Inoltre, La co-presenza di alghe produttrici di palitossine e acido okadaico o loro analoghi, comporta una possibile contaminazione dei prodotti ittici, con effetti tossici nell’uomo conseguenti ad una co-esposizione ad entrambe le tossine e possibili effetti sinergici. Lo scopo della mia ricerca è stato pertanto quello di studiare la tossicità nel topo, acuta e a breve termine, conseguente alla co-esposizione orale alla palitossina e acido okadaico, in confronto a quella delle singole tossine, al fine di individuare gli effetti tossici principali e gli organi bersaglio, ma anche anche un NOAEL (No Observed Adverse Effect Level), da utilizzare per la valutazione del rischio subcronico, verosimilmente lo scenario più rispondente all’esposizione umana. Gli studi sono stati condotti su topi CD-1 di sesso femminile (18-20 g di peso corporeo, 4 settimane; Harlan Laboratories; S. Pietro al Natisone, Udine, Italia). Tutti gli esperimenti sono stati condotti presso l'Università di Trieste in conformità con il Decreto Italiano n. 116/1992, nonché la Direttiva 2010/63/UE dell'UE e la Convenzione europea ETS 123. Inizialmente, sono stati studiati gli effetti indotti dalla somministrazione acuta della palitossina (30, 90 e 270 µg/kg) in associazione con l’acido okadaico (370 µg/kg), rispetto a quelli indotti dalle stesse dosi delle singole tossine. In seguito alla somministrazione intragastrica delle tossine o del veicolo (soluzione salina tamponata con fosfato contenente l’1.8 % di etanolo; controlli) a gruppi di 8 topi, gli animali sono stati monitorati per 24 ore (5 topi/dose) o 14 giorni (3 topi/dose), registrando i sintomi e la sopravvivenza. Dopo il decesso o il sacrificio, è stato effettuato l’esame necroscopico, prelevando campioni ematici per l’analisi ematochimica ed i principali organi e tessuti per l’analisi istologica al microscopio ottico. Entro 24 ore dalla somministrazione, solo nei topi trattati con PLTX alle dosi maggiori (90 o 270 g/kg), singolarmente o con AO (370 g/kg), sono stati rilevati effetti tossici anche letali (letalità: 2/5 topi, in ogni gruppo). In seguito, effetti letali si sono verificati solo alla dose più alta di PLTX, singolarmente (1/3 topi, giorno 9) o con AO (2/3 topi, giorni 4 e 5). I sintomi ed i segni di tossicità (“scratching”, piloerezione, gonfiore addominale, atassia, paralisi agli arti inferiori e dispnea) erano lievemente più intensi nei topi trattati con entrambe le tossine rispetto a quelli dei topi trattati con la sola PLTX. Entro 24 ore dalla somministrazione della PLTX (90 o 270 g/kg), da sola o con l’AO, l’esame necroscopico ha rivelato arrossamento ed accumulo di liquido nell’intestino tenue ed un aumento ponderale del fegato, mentre l’analisi istologica ha evidenziato alterazioni gastriche (lievi ulcerazioni ed infiammazione allo stomaco non ghiandolare) ed epatiche (riduzione di glicogeno negli epatociti). Durante tutto il periodo di osservazione, nei topi trattati con le dosi più alte di PLTX, anche in combinazione con l’AO, sono stati inoltre rilevati un lieve calo del peso corporeo ed una lieve riduzione del consumo di cibo. Negli animali sopravvissuti fino a 14 giorni dal trattamento non sono state invece osservate alterazioni di rilievo. Lo studio ha così evidenziato un lieve effetto additivo tra PLTX ed AO dopo somministrazione acuta per via orale, stimando dei valori di NOAEL pari a 30 g/kg per la PLTX e 370 g/kg per l’AO, singolarmente o in combinazione. Vista la mancanza di studi di tossicità della PLTX dopo ripetute somministrazioni orali, prima di valutare gli effetti della sua somministrazione ripetuta con l’AO, è stato condotto uno studio di tossicità ripetuta sulla sola PLTX. Questa è stata somministrata a gruppi di 6 o 8 topi, una volta al giorno per 7 giorni, a quattro dosi (3, 30, 90 e 180 µg/kg/die). Gli animali sono stai osservati fino a 24 ore dall’ultimo trattamento e, sottogruppi di 3 topi, fino a 14 giorni. Il trattamento ha causato effetti letali e/o tossicità a partire dalla dose di 30 μg/kg/die, dal terzo giorno e, in alcuni casi, il decesso si è verificato durante il periodo di “recovery” in assenza di trattamento, indicando che gli effetti non sono totalmente reversibili. A tali dosi, durante il trattamento sono stati rilevati un significativo calo del peso corporeo, gonfiore addominale, cromodacriorrea, piloerezione, dispnea, sedazione e/o paralisi agli arti posteriori. Alterazioni macroscopiche gastrointestinali (ulcere gastriche e presenza di fluido intestinale) sono stati osservati nei topi morti durante il periodo di trattamento, mentre l’analisi istologica ha rilevato un’infiammazione polmonare, localmente associata a necrosi, iper-eosinofilia e separazione delle fibre nel miocardio ed alterazioni epatiche (ridotto contenuto in glicogeno e necrosi alla dose maggiore). Dai dati ottenuti è stato possibile calcolare un NOAEL “provvisorio” pari a 3 μg/kg/die, indicando una relazione dose-effetto piuttosto ripida. Sono stati quindi studiati gli effetti di 7 giorni di somministrazione con PLTX (3 e 10 g/kg/die) ed AO (185 g/kg/die), osservando che entrambe le dosi di PLTX associate con l’AO hanno causato effetti letali (1/8 topi a 3 g/kg/die ed 1/8 a 10 g /kg/die di PLTX in combinazione con l’AO/kg, nei giorni 7 e 8). Negli stessi gruppi, durante il periodo di trattamento gli animali avevano manifestato “scratching”, dispnea, paralisi agli arti posteriori e calo del peso corporeo. A 24 ore dall’ultimo trattamento, l’analisi necroscopica ha inoltre rivelato un accumulo di fluido nell’intestino tenue dei topi trattati con la dose maggiore di PLTX ed AO, accompagnato da un significativo calo ponderale del fegato. Un effetto a livello epatico è stato evidenziato anche da un incremento delle transaminasi sieriche e, in un topo, da lievi focolai di necrosi epatica, accompagnata da atrofia del timo. A 14 giorni dalla fine del trattamento non sono state invece rilevate significative alterazioni, eccetto un calo del peso corporeo degli animali trattati con l’associazione di AO e PLTX ad entrambe le dosi. Lo studio ha pertanto rivelato un potenziamento della tossicità da co-esposizione orale di PLTX ed AO, in grado di determinare effetti letali ed alterazioni non rilevabili dopo somministrazione delle singole tossine. I risultati complessivi hanno evidenziato che la presenza concomitante delle due tossine, benchè chimicamente differenti, potrebbe aumentare il loro profilo di tossicità esercitanto effetti latenti; anche se non sono stati osservati chiari effetti sinergici o additivi, questi non possono essere completamente esclusi. La caratterizzazione del rischio dovrebbe progredire con studi aventi un gruppo più ampio di animali ed un trattamento prolungato a 14/28 giorni ed oltre. Questi studi, mimando le condizioni alle quali avvengono le intossicazioni umane dovranno includere specifiche valutazioni volte ad indagare l'impatto del fattore età, del deterioramento del tratto gastrointestinale, della variabilità inter-individuale nonchè la possibile attività mutagena delle tossine. Inoltre, l’attuale legislazione dovrebbe essere rivista alla luce della reale quantità ingerita di molluschi da parte dei consumatori, sottolineando l'importanza della valutazione di dosi che producano intossicazioni non clinicamente evidenziabili, come effettuato in questi studi.
XXVI Ciclo
1978

2011/2012
SUMMARY Biotoxins produced by harmful algae during their proliferation can be accumulated by filter feeding organisms, such as bivalve shellfish, within their flesh. Furthermore, these toxins gradually are transferred to the higher trophic levels in the food chain, posing a threat to human health, after consumption of contaminated seafood. Filter-feeding invertebrates are organisms in which the toxin accumulation is a well-known phenomenon, especially during harmful algal blooms. Mussels, cockles, oysters, and scallops feed on toxic dinoflagellates, transferring them from the gills to digestive organs where the toxins accumulate. Different algal toxins can simultaneously contaminate edible shellfish, representing a world-wide sanitary and economic problem. Among them, Palytoxin (PLTX) is a highly toxic polyhydroxylated compound associated to human seafood intoxications in tropical and subtropical areas, but recently it has been detected also in microalgae and shellfish from temperate areas, as Mediterranean Sea. In the last years, also Yessotoxin (YTX) was frequently detected in mussels from Mediterranean Sea and a possible co-exposure to both PLTX and YTX can occur through contaminated seafood consumption. Therefore, the research was initially focused on the detection and quantification of PLTX and YTX in mussels collected in the Gulf of Trieste in order to verify the simultaneous shellfish contamination by these toxins and, subsequently, to study their toxic effects after simultaneous oral exposure. Analyses by liquid chromatography coupled mass spectrometry (LC-MS) did not reveal the presence of PLTX in mussels but identified the presence of YTX together with the diarrheic toxins okadaic acid (OA) and its acyl esters. Consequently, the final goal of this part of the research was the determination of these toxins in mussels from the Gulf of Trieste by LC-MS as well as that of OA and its esters by a functional assay, the protein phosphatase 2A (PP2A) inhibition assay (comparing the results obtained from two different protocols for the PP2A assay). These assays were used to analyse both cooked and uncooked mussel samples, to verify the influence of the heating procedure on the toxin content of mussels. Globally, no significant difference in toxins concentration between uncooked and cooked mussels was observed. However, comparing the data of single samples, a slight increase (not statistically significant) of toxin concentrations was detected in the cooked mussels with respect to the uncooked ones. The mussel analysis by LC-MS/MS detected also the presence of significant amounts of yessotoxin. Contrary to OA group toxins, yessotoxin was slightly less concentrated in the cooked mussels, probably because of its higher polarity that allows a dissolution in the water lost during the cooking procedure. In the other hand and as it was mentioned before, the palytoxin produced by Ostreopsis sps. microalgae have become a problem in more occasions for the attendance of the coastal environment, both for recreation and for business, representing a loss, as well as to public health, even for the tourism and the aquaculture industry. World market globalization, climatic changes and increasing overseas traffic are considered the main responsible for the appearance of these toxins. The expand of these toxins to temperate regions may be due in part to ballast water of ships and also to general changes in climate conditions, enough to induce bloom formation. Despite the extent of the contaminated area, few methods are currently available for palytoxins detection and quantitation in seafood. Moreover, among palytoxins, only palytoxin is commercially available, though expensive, and no certified standard material is currently sold. For monitoring purposes, a combination of screening methods followed by a chemical confirmatory analysis, such as LC-MS, is commonly used to detect palytoxins. Other methods for PLTX analysis include mouse bioassay, cytotoxicity assays, haemolysis assays, receptor binding assays, and immunoassays. Even if there were no food poisoning from palytoxin in the Mediterranean countries, the toxin was detected in shellfish, which gave positivity to the official test for the lipophilic toxins of algal origin (okadaic acid and derivatives, azaspiracids, yessotoxins and pectenotoxins). Some different experiments were applied in the evaluation of palytoxin toxicity in this study (Haemolytic assay, sandwich ELISA, LC-MS/MS and cytotoxicity studies were carried out). Haemolytic assay, carried out incubating mouse erythrocytes with palytoxin for 4 h (standard assay) or for 1 h in diluted PBS (abbreviated assay) is able to detect palytoxin at picomolar concentrations. Nevertheless, with the aim to detect the toxin in mussels, a significant matrix effect impairing the toxin quantification was observed already at the mussel extract concentration of 0.048 mg edible tissues equivalents/mL, which was more evident by the abbreviated assay. However, most of the experiments lack specificity or have other limitations. Thus, an indirect sandwich ELISA has been set up. The ELISA assay (indirect sandwich) was developed using the monoclonal antibody 73D3, and a rabbit polyclonal antibody produced at the University of Trieste. The assay detects the PLTX in a range of concentrations ranging from 1.25 to 40 ng/ml and is able to quantify with very similar sensitivity also biotinilated PLTX as well as 42-OH-PLTX, this latter isolated and characterized from the chemical point of view during the latter years from the group of prof. E. Fattorusso (University of Naples Federico II), in a sample of palytoxin kindly provided by Dr. M. Poly (Maryland, USA). The incapacity to detect okadaic acid (OA), domoic acid (DA), brevetoxin-3 (PbTx-3), saxitoxin (STX) and yessotoxin (YTX)(toxins that may be present along with PLTX in fish contaminated) indicates the specificity of the assay. The structure of Palytoxins is very complex. In addition to this structural complexity, there is still a lack of knowledge about the different congeners involved in this contamination and therefore there is still a very limited availability of standards and reference materials. These issues made difficult the advances in the development and optimization of analytical methods, particularly in the case of LC-MS/MS. Despite of this, a significant progress has been made over the last few years in the development of analytical techniques, particularly on LC-MS/MS approaches. In this part of the study, a LC-MS/MS method was optimized for the analysis of PLTXs in order to be able to detect, quantify and confirm the presence of this toxins in natural samples. During the PhD period, there was the possibility to get some natural contaminated samples to be evaluated by ELISA developed assay and then compare the results with the analysis by the developed LC-MS/MS method. The study was carried out with samples from 3 distinct sites characterized by having different coastal morphologies and continental hydrodynamic conditions: i) Madeira Islands’ archipelago in the NE Atlantic Ocean, Selvagens island in particular (Long, Lat) during the upwelling of August 2008; ii) Cascais, on the west coast of Portugal mainland, located at the northern side of Lisbon bay during the upwelling occurred during the favourable northerly wind periods (from April to September 2011); and iii) Algarve, Lagos, on the South Portuguese coast, also in 2011.
RIASSUNTO Biotossine prodotte da alghe nocive durante la loro proliferazione possono essere accumulati da organismi di alimentazione per filtrazione, come molluschi bivalvi, nell'ambito della loro carne. Inoltre, queste tossine vengono trasferite gradualmente ai livelli trofici superiori della catena alimentare, che rappresenta una minaccia per la salute umana, dopo il consumo di frutti di mare contaminati. Invertebrati con filtro-alimentazione sono organismi in cui l'accumulo di tossine, è un fenomeno ben noto, soprattutto durante fioriture algali nocive. Cozze, vongole, ostriche e capesante si nutrono di dinoflagellati tossici, trasferendoli dalle branchie agli organi digestivi, dove le tossine si accumulano. Diverse tossine algali possono contemporaneamente contaminare molluschi commestibili, che rappresenta un problema mondiale sanitario ed economico. Tra questi, Palitossina (PLTX) è un composto altamente tossico poliossidrilato associato ad intossicazioni ittici dell'uomo nelle zone tropicali e subtropicali, ma recentemente è stato rilevato anche in microalghe e molluschi dalle zone temperate, come il Mare Mediterraneo. Negli ultimi anni, anche yessotossina (YTX) è stato spesso rilevato nei mitili dal Mare Mediterraneo e una possibile co-esposizione sia PLTX e YTX può avvenire attraverso il consumo di frutti di mare contaminati. Pertanto, la ricerca è stata inizialmente concentrata sul rilevamento e la quantificazione di PLTX e YTX nei mitili raccolti nel Golfo di Trieste, al fine di verificare la contaminazione simultanea nei frutti di mare da queste tossine e, in seguito, per studiare i loro effetti tossici dopo esposizione orale simultanea. Analisi mediante cromatografia liquida accoppiata alla spettrometria di massa (LC-MS) non ha rivelato la presenza di PLTX in mitili ma è stata identificata la presenza di YTX insieme alle tossine diarroiche acido okadaico (AO) e suoi esteri. Di conseguenza, l'obiettivo finale di questa parte della ricerca è stata la determinazione di queste tossine nei mitili del Golfo di Trieste mediante LC-MS così come quella di AO e suoi esteri da un saggio funzionale, il Saggio d’inibizione Proteina Fosfatasi 2A (PP2A)(confrontando i risultati ottenuti da due diversi protocolli per il saggio PP2A). Questi test sono stati utilizzati per l'analisi di campioni di mitili sia crude e cotte, per verificare l'influenza della procedura di riscaldamento sul contenuto di tossina nei mitili. A livello globale, nessuna differenza significativa nella concentrazione di tossine tra cozze crude e cotte è stata osservata. Tuttavia, confrontando i dati dei singoli campioni, un lieve aumento (non statisticamente significativo) delle concentrazioni di tossine è stato rilevato nelle cozze cotte rispetto a quelli crude. L'analisi dei mitili per LC-MS/MS rilevò anche la presenza di quantità significativa di yessotossina. Contrariamente alle tossine gruppo OA, yessotossina era leggermente meno concentrata nelle cozze cotte, probabilmente a causa della sua polarità superiore che permette una dissoluzione in acqua persa durante la cottura. Altrimenti e come è stato detto prima, la palitossina prodotta da Ostreopsis sps. microalghe sono diventate un problema in più occasioni per la partecipazione dell'ambiente costiero, sia per la ricreazione e per le imprese, con una perdita, così come per la salute pubblica, anche per il turismo e l'industria dell'acquacoltura. Globalizzazione del mercato mondiale, i cambiamenti climatici e l'aumento del traffico all'estero sono considerati il principale responsabile della comparsa di queste tossine. L'espansione di queste tossine per le regioni temperate può essere dovuto in parte alla acque di zavorra delle navi e anche a cambiamenti delle condizioni climatiche generali, tanto da indurre la formazione di fioritura. Nonostante e dovuto alla estensione dell'area contaminata, alcuni metodi sono disponibili per il rilevamento e la quantificazione di palitossina in frutti di mare. Inoltre, tra palitossine, solo palitossina è disponibile in commercio, anche se costoso, e nessun materiale standard certificato è attualmente venduto. A scopo di monitoraggio, una combinazione di metodi di screening seguita da una analisi chimica di conferma, ad esempio LC-MS, è comunemente utilizzato per rilevare palitossine. Altri metodi di analisi includono PLTX biotest sui topi, saggi di citotossicità, saggi emolici, saggi di legame al recettore e saggi immunologici. Anche se non ci sono stati identificate intossicazione alimentare da palitossina nei paesi del Mediterraneo, la tossina è stata rilevata nei molluschi, che ha dato positività al test ufficiale per le tossine lipofile di origine algale (acido okadaico e derivati, azaspiracidi, yessotossine e pectenotossine). Alcuni esperimenti diversi sono stati applicati nella valutazione della tossicità della palitossina in questo studio (saggio emolitico, ELISA, LC-MS/MS e studi di citotossicità sono state effettuate). Saggio emolitico, effettuato incubando eritrociti di topo con palitossina per 4 h (saggio standard) o per 1 h in PBS diluito (saggio abbreviato) è in grado di rilevare la palitossina a concentrazioni picomolari. Tuttavia, con lo scopo di rilevare la tossina nelle cozze, un significativo effetto matrice ledere la quantificazione di tossina è stata osservata già alla concentrazione di estratto di cozze 0,048 mg equivalenti tessuti commestibili/mL, che è stato più evidente con il saggio abbreviato. Tuttavia, per la maggior parte degli esperimenti mancano specificità o hanno altre limitazioni. Così, un indiretto sandwich ELISA è stato istituito. Il saggio ELISA (sandwich indiretto) è stato sviluppato utilizzando gli 73D3 anticorpi monoclonali, e un anticorpo policlonale di coniglio prodotto nella Università di Trieste. Il saggio rileva la PLTX in un intervallo di concentrazioni variabili 1,25-40 ng / ml ed è in grado di quantificare con sensibilità molto simile anche PLTX biotinilata così come 42-OH-PLTX, quest'ultimo isolata e caratterizzata dal punto di vista chimico durante gli ultimi anni dal gruppo del prof. E. Fattorusso (Università degli Studi di Napoli Federico II), in un campione di palitossina gentilmente fornito dal Dr. M. Poli (Maryland, USA). L'incapacità di individuare acido okadaico (AO), acido domoico (AD), brevetossina-3 (PbTx-3), saxitossina (STX) e yessotossina (YTX) (tossine che possono essere presenti insieme a PLTX nel pesce contaminato) indica la specificità del dosaggio. La struttura della palitossina è molto complessa. In aggiunta a questa complessità strutturale, vi è ancora una mancanza di conoscenza sui diversi congeneri coinvolti in questa contaminazione e quindi c'è ancora molto limitata disponibilità di standard e materiali di riferimento. Questi problemi reso difficili gli progressi nello sviluppo e ottimizzazione di metodi analitici, in particolare nel caso di LC-MS/MS. Nonostante, un progresso significativo è stato compiuto negli ultimi anni allo sviluppo di tecniche analitiche, in particolare su approcci LC-MS/MS. In questa parte dello studio, un metodo LC-MS/MS stato ottimizzato per l'analisi di PLTXs per essere in grado di rilevare, quantificare e confermare la presenza di queste tossine in campioni naturali. Durante il periodo di dottorato di ricerca, c’è stata la possibilità di ottenere alcuni campioni naturali contaminati da valutare tramite il saggio ELISA sviluppato e poi confrontare i risultati con l'analisi con il metodo sviluppato di LC-MS/MS. Lo studio è stato effettuato con campioni da 3 posti diversi caratterizzati d’avere diverse morfologie e condizioni idrodinamiche costiere continentali: i) arcipelago Isole Madeira nel nord-orientale dell'Oceano, Selvagens isola in particolare durante il mese di agosto 2008; ii) Cascais, sulla costa occidentale del Portogallo continentale, che si trova sul lato settentrionale della baia di Lisbona durante i periodi favorevoli di vento dal nord (da aprile a settembre 2011), e iii) Algarve, Lagos, sulla costa sud-portoghesa, anche nel 2011.
XXV Ciclo
1983

碩士
國立臺灣海洋大學
食品科學系
103
Palytoxin (PLTX) and analogs (PLTXs) are a group of the most dangerous marine toxic compounds, which could be accumulated in marine organisms, such as Palythoa spp., Ostreopsis spp., crab and several fish species. PLTXs are considered as the causative agent for human seafood poisoning with symptoms of nausea, vomit, gastro-intestinal malaise, myalgia, muscular cramps, dyspnea and arrhythmias. People would get sick or even dead by ingesting PLTXs. The poisoning accidents of PLTXs happened all around the world. In this research, the zoanthids collected from Green Island, Taiwan were identified as Palythoa tuberculosa. By ethanol extraction and ion exchange column chromatography, the purity of PLTXs was improved. After the purification of PLTX from P. tuberculosa extracts, high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometer (LC-MS/MS) were used to identify the toxic compounds compared with PLTX standard. The purified extract was mainly detected as palytoxin. Moreover, mouse bioassay was utilized to detect the toxicity of purified extract. The death times of mice were recorded after intraperitoneal injection, and the median lethal dose (LD50) was 1.51 µg/ml (7.55 µg/kg b.w.). Furthermore, the histological analysis revealed vacuolation and necrosis in liver and kidney. Hematoclinical analysis were attempted by detection of creatine phosphokinase (CPK), aspartate aminotransferase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), K+and Na+. The results showed increasing levels of ALT, AST, CPK and LDH after 4 hr reaction time, and the tendency of increase as dose increased. Also, the levels of K+ was increased as dose and reaction time increased, but the levels of Na+ was not significant different. The haemolytic activity was suppressed after 4 hr by pretreatment with 1 mM ouabain for 1 hr.

碩士
國立臺灣海洋大學
食品科學系
99
Palytoxin is one of the most potent marine toxic compound with non-protein structure, and it causes food poisoning cases by food-chain accumulation. In Pacific area, trunkfish is one kind of food. Especially in Japan, many palytoxin-like food poisoning cases occurred by ingestion of trunkfish. It also occurred once in Taiwan; however, palytoxin was not identified in this case until now. In this study, we first studied on palytoxin-like toxicity from family Ostraciidae fish in Taiwan by hemolysis activity and mouse bioassay. The fish seem to contain palytoxin-like compounds. In aged trunkfish, the toxicity in liver tissue is more toxic than that in muscle, but it is opposite in smaller trunkfish. In Taiwan, toxic dinoflagelate Ostreopsis spp. And soft coral Palythoa spp. are the main resources of palytoxin. But the toxin profiles of palytoxin in Taiwan is still lack. In the second part, we used hemolysis activity of palytoxin to investigate palytoxin stability under different ion, acid and alkali conditions. The results showed that hemolysis activity of palytoxin was significantly reduced when treated with Cu2+ and acetic acid (pH 3). Fe3+, Fe2+ and Zn2+ would slightly inhibit the hemolysis activity of palytoxin. Hydrogen chloride, sodium hydroxide and sodium carbonate would not change the hemolysis activity of palytoxin.

碩士
國立臺灣海洋大學
食品科學系
106
Palytoxin (PTX) and analogs (PTXs) are a group of the most potent marine toxins produced by some zoanthids of the genus Palythoa and Zoanthus, dinoflagelllate Ostreopsis and cyanobacteria Trichodesmium. They can be also accumulated in marine organisms via the food chain. People who consume these contaiminated seafood have the risk of food poisoning. Taiwan is located in the subtropical area and has the producer of toxin.There also has been reported one case of human intoxication resulting a fatal outcome. Therefore, we should faced up to the hazard that the toxin may be harmful to human and tried to establish the relevant toxicity information. In this study, the gene sequences of zoanthid samples which collected from the Da-Ping coast of Keelung, Taiwan were analyzed by polymerase chain reaction (PCR) technique with direct sequencing. All of the samples were identified as the same species, Palythoa tuberculosa. Then the toxicity of the samples in each month was first detected by mouse bioassay. After the mice were injected with the extracts of samples collected in August and October, the mice showed PTXs symptoms such as stretching of hind limbs, diarrhea, and loss of appetite. In addition, the extracts also have the ability to increase the creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activities in mouse serum. On the other hand, hemolytic activity was detected in the extracts in August and October, which were 10.00% and 25.69%, respectively. The biological activities of above three indicators showed that the extracts of P. tuberculosa from Keelung,Taiwan contained PTXs, and its toxicity was more stronger in August and October. In order to understand the toxic effects of PTXs on vascular endothelial cells (SVEC 4-10), another zoanthid sample (P. tuberculosa) collected from Green Island,Taiwain were used for cytotoxicity test. The results of MTT assay showed that the extract (1-25 μg/mL) is toxic to SVEC4-10 cells and can change the cell morphology from polygonal to spherical. However, the cytotoxicity was inhibited after cells pretreated with ouabain. It was further suggested that the extract contained PTXs. In this experiment, the extracts could induce several adverse effects such as elevating generation of reactive oxygen species (ROS) and nitric oxide (NO), decreasing total thiol content, and reducing mitochondrial membrane potential. The type of cell death induced by the extract was investigated by Annexin V/PI double staining using flow cytometry. It was found that apoptosis (low concentration) and necrosis (high concentration) were all possible forms of cell death depending on the concentration of the extract. These results suggest that PTXs may cause increased intracellular oxidative stress, lead to mitochondrial dysfunction and finally induce cell death.

碩士
國立臺灣海洋大學
食品科學系
101
Palytoxin (PTX) and analogs (PTXs) are a group of the most dangerous marine toxic compounds with non-protein structure, which could be accumulated in marine organisms. PTXs have been detected in crabs and several species of fishes. People would get sick or even dead if they had eaten marine organisms containing PTXs. The frequent symptoms of PTXs poisoning are including nausea, vomit, dizziness, diarrhea and breathing difficulties. In addition, PTXs could cause abnormal rise of serum creatine phosphokinase (CPK). Every year, poisoning accidents happened all around the world, as eating the marine organisms by mistake. Hence, PTXs toxicity and the detection methods in this study are very important. In the first chapter, poisoning accident occurred in Taitung, Taiwan was investigated. Two persons, who caught and ate the poisoning sample, felt uncomfortable and one of them died in the end. By mouse bioassay, the death times of mice were recorded after intraperitoneal injection of fish liver and muscle extraction. High concentration of toxic compound was speculated in the fish sample. Elevated level of CPK was also observed after intraperitoneal injection. Hemolytic activity of fish extraction was observed too. In order identify the toxic compound, fish liver and muscle extracts were analyzed as PTXs by high performance liquid chromatography (HPLC). Finally, the DNA sequences of fishes were compared with the data base in NCBI, and the causative fish is identified as Herklotsichthys sp. In the second chapter, DNA sequence of zoanthids collected from Green Island, Taiwan and Ishigaki, Japan were identified in the second chapter. The zoanthids collected from these two places belong to the same species Palythoa tuberculosa. Compared with PTXs standard by HPLC, PTXs actually exist in sample collected from Green Island. In order to make sure that the PTX standard is not degraded, the PTX standard was analyzed with liquid chromatography - mass spectrometer (LC-MS), and identified as three different structures of PTXs (palytoxin, 42-hydroxy-palytoxin and deoxy-palytoxin). By mouse bioassay, the death times of mice were recorded after intraperitoneal injection of zoanthid extraction, and median lethal dose (LD50) is calculated as 116.3 μg/ml. Elevated level of CPK was observed by PTXs mouse bioassay. Besides, hemolytic activity of PTXs was observed by testing the erythrocyte of rats. Half maximal effective concentration (EC50) of PTXs hemolysis was defined as 324.21 μg/ml.