Palytoxin and Okadaic acid as seafood contaminants: risk caracterization.

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.