扇贝滤食甲藻的过程中麻痹性贝毒代谢轮廓及代谢物毒性研究

There has been an increasing occurrence of toxic algal blooms world-wide, along with increased risk to human health due to contamination of seafood with toxins. The issue of seafood safety is particularly critical with the deadly paralytic shellfish toxins (PST). Currently, there is only a partial understanding of the metabolic transformation, toxicity and depuration of PST in bivalves, yet it is very important to understand all the structural forms and toxicities of these toxins in our food in order to protect consumer health. Recently, the structures of five PST metabolites (M1-5) have been published and our preliminary research has revealed the presence of several other structural analogs. These metabolites had been missed in many in vivo or in vitro experiments for biotransformation of PST in shellfish in which only familiar toxin components were studied. In the present grant application, these new metabolites will be focused on as targets in different tissues and depuration water of two species of scallop, including Patinopecten yessoensis　and　Chlamys farreri, fed with PST-producing dinoflagellates, Alexandrium tamarense and A. minutum. A comprehensive search for new metabolites and their identification by LC-MS/MS will be conducted. Based on toxin transformation data and distribution in different tissues of scallop, some intermediate products of "phase II metabolism" will be also explored by comparing the mass chromatograms of toxic tissues with those of non-toxic tissues in scallop. Accurate reference materials of metabolites, particularly M1,M3,and M9, will be produced after their isolation and purification from toxic shellfish, using column chromatography and semi-preparative chromatography. Toxicity of these isolated metabolites will be quantified by the "patch clamp technique" developed using HEK 293 cells. Stability of reference materials stored at different temperature and pH conditions will be also assessed in the laboratory. The dynamic concentrations of all toxin components and metabolites will be monitored in gill, hepatopancreas, kidney, adductor muscle, and blood of scallop during toxin-accumulating periods using LC-MS/MS. Toxin components depurated from scallop contaminated by PST will be characterized using the solid phase adsorption toxin tracking (SPATT) technique and LC-MS/MS. The objective of this proposal is a comprehensive interpretation of the metabolic profile of PST and their contribution to the total toxicity in shellfish. This study will contribute to the development of toxin "metabolomics" in shellfish. This is very important for supporting the sustainable development of shellfish sea-culture and the protection of consumer health.

麻痹性贝毒（PST）业已成为影响贝类水产品安全的重要因子，潜在威胁人类健康和海洋生态安全。近年研究发现PST染毒贝类普遍含有高浓度的C11位羟基代谢物（如M1-5），但以往的工作缺少对这类代谢物的分析，亟待深入研究。本项目通过分离纯化贝类中含量较高的主要代谢物（M1、M3和M9），制备一定量的标准品并确定其毒性和稳定性。选择虾夷扇贝和栉孔扇贝为研究对象，模拟扇贝滤食塔玛亚历山大藻、微小亚历山大藻的染毒及不喂食条件下的排毒过程，定量分析扇贝的鳃、肝胰腺、肾脏、闭壳肌和血液等不同组织内PST常见组分及代谢物含量的动态变化，监测染毒扇贝排毒过程的毒素组成及排毒速率，并探索扇贝体内PST代谢可能存在的"第Ⅱ相反应"途径。以期阐释PST在扇贝体内的代谢轮廓及其代谢物对扇贝总毒性的影响，为贝类毒素"代谢组学"的研究奠定基础，对保障贝类养殖经济的可持续发展和保护消费者健康具有重要意义。

麻痹性贝毒（paralytic shellfish toxins，PST）是一类急性毒性强、分布范围广、消费者中毒风险高的海洋藻毒素。该毒素的产毒甲藻在我国沿海广泛分布，近两年分别在河北省和福建省近海引发消费者中毒事件，严重威胁人体健康和海洋生态安全。本项目以近年新发现的PST在贝类体内的C11位羟基代谢物（M-toxins）为目标，采用室内模拟栉孔扇贝和紫贻贝滤食产毒藻的染毒实验，监测贝类体内M-toxins的产生与排出过程，并结合野外采集的PST染毒贝类样品的毒素分析结果，揭示了M-toxins在贝类体内的主要代谢转化途径。结果表明M1来自贝类积累的C2毒素，同时M1可以产生代谢物M3，并推测M2、M4的产生与GTX2/3有关，相应地M7、M9来自C4毒素，M8、M10的产生与GTX1/4有关；且C2和GTX2/3毒素向代谢物转化的速率分别明显高于C4和GTX1/4毒素。通过高分辨质谱对比分析贝类染毒前后样品的全扫描母离子的二级质谱以及对假定的几种PST“Ⅱ相反应”的结合产物的分析，均未发现与PST结构相关的结合产物，说明PST这种极性较强的毒素可能在贝类体内不具有“Ⅱ相反应”的代谢途径。采用两种不同内径尺寸的Bio-Gel P2树脂填充的层析柱，对贝类滤食甲藻染毒的样品进行分离纯化，初步获得了一定量的较高纯度的代谢物制品，并采用受体结合分析法初步验证了其毒性与C类毒素相似。本项目的研究成果提高了人们对贝类体内PST代谢途径的认识，为分离纯化PST代谢物标准品积累了重要经验，为贝类毒素“代谢组学”的研究奠定了基础，对保障贝类水产品食用安全和保护消费者健康具有重要意义。