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

我们在野外调查中指出，双壳贝类在缺乏甲藻的情况下，往往会积累PSP毒素。本研究的目的是通过实验室实验重新认识贝类毒素积累的机制。用塔玛亚历山大藻培养细胞对贝类进行饲养实验，结果表明贝类可将积累的PSP毒素释放到环境水体中。但是，也观察到一些奇怪的现象，即：（1）双壳贝类积累的毒素多于供给的毒素;（2）停止毒素供给后，双壳贝类体内积累的毒素与环境水体中积累的毒素之和先下降，然后又上升。这些现象与现场调查中观察到的现象相似。在反应中，其中11-O-硫酸的gonyautoxins（GTX）是还原性去除硫醇，如谷胱甘肽（GSH），毒素和硫醇的稳定共轭物被发现形成。现象2）表明GTX与双壳类的GSH结合形成GS-STXs ...更多信息 其不能通过PSP分析检测，然后毒素再次从缀合物中释放。然而，没有检测到显著的GS-STX。这是由于一系列反应，其中GS-STX被gGTP消化以形成STX与半胱氨酰甘氨酸的缀合物，并且半胱氨酸的氨基将STX分解为类似于毒素的碱处理中的荧光化合物。用抗SIX抗体对有毒扇贝消化腺的薄片进行染色时，阿米巴细胞的胞浆和营养细胞周围的膜被染色，表明毒素与这些组织中的蛋白质结合。通过凝胶过滤从消化腺制备的蛋白质级分通过用巯基乙醇处理得到STX，表明毒素与这些级分中蛋白质的半胱氨酸残基的氨基结合;另一方面，当用蛋白酶消化上述蛋白质级分时，释放GTX，表明一些GTX以与半胱氨酸残基的巯基不同的方式与蛋白质结合。此外，在蛋白酶消化的蛋白质组分中也观察到荧光物质，表明与半胱氨酸残基结合的STX通过蛋白酶消化蛋白质而被分解。这是关于双壳贝类毒素分解的首次发现，其中涉及双壳贝类的酶。本研究结果为PSP毒素代谢的进一步研究提供了线索。少

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