Search

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

Palytoxin (PLTX) is a highly toxic hydrophilic polyether detected in several edible marine organisms from intra-tropical areas, where seafood poisoning were reported. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnea and, sometimes, arrhythmias. Monitoring programs in the Mediterranean Sea have detected PLTX-like molecules in edible mollusks and echinoderms. Despite the potential exposure of the human population and its high toxic potential, the toxicological profile of the molecule is still an issue. Thus, the effects of repeated oral administration of PLTX in mice were investigated. Seven days of PLTX administration caused lethality and toxic effects at doses ≥30 μg/kg/day. A NOAEL was estimated equal to 3 μg/kg/day, indicating a quite steep dose–response curve. This value, due to the limited number of animal tested, is provisional, although represents a sound basis for further testing. Macroscopic alterations at gastrointestinal level (gastric ulcers and intestinal fluid accumulation) were observed in mice dead during the treatment period. Histological analysis highlighted severe inflammation, locally associated with necrosis, at pulmonary level, as well as hyper-eosinophilia and fiber separation in myocardium. A cardiac damage was supported by the in vitro effect of the toxin on cardiomyocytes, indicating a severe and irreversible impairment of their electrical properties: electrophysiological recordings detected a progressive cell depolarization, arrest of action potentials and beating.

The polyethers yessotoxin (YTX) and okadaic acid (OA) are two marine algal toxins frequently associated as edible shellfish contaminants. Seafood contamination by these compounds, also at low concentrations and for a long period of time, can increase the possibility of their simultaneous and repeated ingestion, with possible synergistic toxic effects. Thus, in vivo toxicity by repeated oral exposure to a combination of fixed doses of YTX and OA (1 mg YTX/kg and 0.185 mg OA/kg, daily for 7 days) was investigated in mice, in comparison to that of each toxin alone. No mortality, signs of toxicity, diarrhea or hematological changes was induced by the toxins co-administration or by the single toxins. Light microscopy revealed changes at gastric level (multifocal subacute inflammation, erosions and epithelial hyperplasia) in 2/5 mice co-administered with the toxins. In animals dosed only with OA, epithelial hyperplasia of forestomach and slight focal subacute inflammation of its submucosa were noted. No changes were induced by the treatment with YTX. Ultrastructural analysis of the heart revealed some cardiomyocytes with “loose packing” of myofibrils and aggregated rounded mitochondria in mice co-administered with the toxins or with YTX; OA-treated mice showed only occasional mitochondrial assemblage and dilated sarcomeres. Thus, the combined oral doses of YTX (1 mg/kg/day) and OA (0.185 mg/kg/day) did not exert cumulative or additive toxic effects in mice, in comparison to the single toxins.

The acute oral toxicity of palytoxin (PLTX), a highly toxic compound associated with seafood intoxication in tropical and subtropical areas, was investigated in mice. After gavage administration (300-1697 microg/kg) to groups of five female CD-1 mice, signs of toxicity and lethality were recorded for 24 h. The LD(50) was 767 microg/kg (95% confidence limits: 549-1039 microg/kg) and the main symptoms observed were scratching, jumping, respiratory distress and paralysis. Hematoclinical analyses showed increased levels of creatine phosphokinase and lactate dehydrogenase at doses of 600 microg/kg and above, and aspartate transaminase at 848 microg/kg and above. Histological analysis revealed acute inflammation of the forestomach in mice surviving up to 24h after administration (424-1200 microg/kg). Other histological alterations were observed in the liver and pancreas, while cardiac and skeletal muscle cells revealed only ultrastructural alterations visible by transmission electron microscopy. Ultrastructural and hematoclinical findings suggest an involvement of skeletal and/or cardiac muscle as targets of PLTX, according to the observed human symptoms. A NOEL of 300 microg/kg can be estimated from this acute oral toxicity study.