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

贝类积累麻痹性贝毒毒素机制的再思考

基本信息

批准号：
10460096
负责人：
KODAMA Masaaki
金额：
$ 7.36万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

项目摘要

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

我们在野外调查中指出，双壳类在没有致病甲藻的情况下，往往会积累PSP毒素。本研究的目的是通过实验室实验来重新认识双壳类毒素积累的机制。塔玛亚历山大藻培养细胞对双壳贝类的饲养实验表明，双壳贝类向环境水体中释放了积累的PSP毒素。然而，观察到了奇怪的现象，即1)双壳类积累的毒素多于供应的毒素，2)停止毒素供应后，双壳类体内的累积毒素和环境水中的毒素总和一度减少，然后又增加。这些现象与实地调查中观察到的现象相似。在用谷胱甘肽(GSH)等硫醇还原脱除藻毒素(GTX)的11-O-硫酸盐的反应中，发现毒素与硫醇形成稳定的结合物。这一现象表明GTXs与双壳类的谷胱甘肽结合形成GS-STXs…更多的是PSP分析无法检测到的，然后毒素再次从结合物中释放出来。然而，没有检测到显著的GS-STX。这是由于GS-STXs被GGTP消化形成半胱氨酸甘氨酸偶合物，半胱氨酸氨基将STXs分解成类似于碱处理毒素的荧光化合物的一系列反应。用抗SiO_6抗体对扇贝有毒消化腺薄片进行染色时，可见阿米巴细胞胞浆和营养细胞周围膜染色，表明毒素与这些组织中的蛋白质结合。从消化腺经凝胶过滤得到的蛋白质组分经巯基乙醇处理后得到STX，表明毒素与这些组分中蛋白质的半胱氨酸残基的氨基结合；另一方面，当上述蛋白质组分被蛋白酶消化时，GTXs被释放，表明一些GTXs与蛋白质结合的方式与半胱氨酸残基中的硫醇不同。此外，在用酶消化的蛋白质组分中也观察到了荧光物质，这表明与半胱氨酸残基结合的STX是通过酶消化蛋白质而分解的。这是关于双壳类毒素分解的第一个发现，双壳类的酶参与其中。本研究结果可为今后PSP毒素代谢的研究提供线索。较少