DEVELOPMENT AND CHARACTERIZATION OF NEW METHODS FOR PALYTOXIN DETECTION

Palytoxin (PLTX) and its analogs are toxic marine polyethers detected in Palythoa corals, Ostreopsis dinoflagellates and Thricodesmium cyanobacteria. The main concern for human health related to these toxins is their accumulation in marine edible organisms and the possible entrance in the food chain up to humans. In tropical areas, human cases of even fatal foodborne poisonings had been ascribed to consumption of PLTXs contaminated seafood. On the other hand, in the Mediterranean Sea, human poisonings ascribed to PLTXs were associated to inhalation of marine aerosol and/or cutaneous exposure to seawater during Ostreopsis blooms and no foodborne poisoning associated with these toxins were documented in this area, so far. Although PLTXs are not regulated, the European Food Safety Authority (EFSA) suggested a maximum limit of 30 µg PLTXs/kg of shellfish meat, recommending the development of suitable, specific and rapid detection methods in seafood. Moreover, given the growing cases of adverse effects during Ostreopsis blooms in the Mediterranean Sea, the development of methods for PLTXs detection also in microalgae is suggested. Thus, two methods for PLTXs quantitation were developed and characterized: a hemolytic assay, based on PLTXs ability to convert the Na+/K+ ATPase into an unspecific cationic channel leading to erythrocytes lysis, and a cell-based immunoenzymatic assay (ELISA), measuring the binding of PLTXs to Na+/K+ ATPase of HaCaT cells by a mouse monoclonal anti-PLTX antibody targeted by an enzyme-conjugated anti-mouse detection antibody. The limits of PLTX detection (LOD) and quantitation (LOQ) by hemolytic assay were 1.4x10-10 M and 3.4x10-10 M, respectively, while those by the cell-based ELISA were 1.2x10-11 M and 2.8x10-11 M, respectively. The two methods were assessed for their suitability to quantify PLTXs in mussels (Mytilus galloprovincialis) and microalgae. The LOQ for PLTX in mussels by the hemolytic assay was 650 µg/kg meat, a value about 20 times higher than the limit suggested by EFSA (30 µg PLTXs/kg meat). On the other hand, the LOQ for PLTX in mussels by the cell-based ELISA was 9.3 µg/kg meat, a value below the safety limit suggested by EFSA. The high sensitivity of the cell-based ELISA, make this method suitable for PLTX detection in monitoring program. The LOQ by the hemolytic assay and the cell-based ELISA in microalgae were 0.03 pg/cell and 0.007 pg/cell, respectively. The second part of this thesis was focalized on the investigation of the sensitivity of different cell models to PLTX on which subsequent studies could be carried out to further characterize PLTX mechanism of action and to develop a new functional detection method for PLTXs. The sensitivity was evaluated by means of cytotoxicity and binding assay. The MTT assay showed Caco-2 cells as the most sensitive to PLTX cytotoxic effect with an EC50 = 3.1x10-11 M (95% C.I. = 5.9x10-12 - 3.4x10-11), while HaCaT cell line was assumed as the most sensitive cell line for PLTX binding, as confirmed by the Bmax and Kd values, equal to 0.043 and 1.4x10-10 M, respectively. The high sensitivity to PLTX demonstrated for Caco-2 cell line by the MTT assay and for HaCaT cell line by the binding assay, was not related to the expression of the Na+/K+ ATPase, suggesting that other factors, such as the expression of specific Na+/K+ ATPase isoforms may play an important role in cells sensitivity to PLTX.