Reconsideration of the mechanism for shellfish to accumulate paralytic shellfish poisoning toxins

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We have pointed out in the field survey that bivalves often accumulate PSP toxins during absence of the causative dinoflagellates. The purpose of the present study is to reconsider the mechanism for bivalves to accumulate toxins by the laboratory experiments. Feeding experiments of cultured cells of Alexandrium tamarense to bivalves showed that bivalves released accumulated PSP toxins to the environmental water. However, curious phenomena were observed, that is, 1) bivalves accumulate toxins more than supplied, 2) After the cessation of toxin supply, the sum of accumulated toxins in bivalve and those in the environmental water once decreased, and then increased again. These phenomena are similar to those observed in the field survey. In the reaction in which 11-O-sulfate of gonyautoxins (GTX) is reductively removed by thiols such as glutathione (GSH), stable conjugates of toxins and thiols are found to form. The phenomenon 2) suggests that GTXs bind with GSH of bivalves to form GS-STXs … More which cannot be detected by PSP analysis, and then the toxins are released from the conjugates again. However, no significant GS-STXs was detected. It was revealed to be due to a series of the reactions in which GS-STXs is digested by gGTP to form the conjugate of STXs with cysteinyl glycine, and the amino group of cystein decomposes STXs to fluorescent compounds similar to those in alkaline treatment of toxins. When the thin section of toxic scallop digestive gland was stained with antibody against SIX, cytosol of amoebarcytes and membranes surrounding nutrient cells were stained, suggesting that toxins are bound with proteins in these tissues. Protein fractions prepared from the digestive gland by gel filtration gave STXs by treatment with mercaptoethanol, showing that toxins are bound with amino group of cystein residues of proteins in these fractions; On the other hand, GTXs were released when the protein fractions described above are digested with protease, showing that some GTXs are bound with proteins in a different manner from that involved in thiol of cystein residue. Furthermore, fluorescent substances are also observed in the digested protein fractions with protease, indicating that STXs bound with cystein residue are decomposed by digestion ofproteins with protease. These are the first finding on the decomposition of toxins in bivalves in which the enzymes of bivalves are involved. The results of the present study could give a clue for the future study on metabolism of PSP toxins. Less