海洋生态与环境科学重点实验室知识库

麻痹性贝毒是一类神经性毒素，主要由海洋中的甲藻产生，可以通过双壳贝类的滤食作用在其体内累积，导致消费者中毒，对人类健康和生命安全具有严重威胁。染毒贝类中的毒素含量和毒性一方面取决于其滤食的产毒藻中毒素组成和含量状况，另一方面也受到贝体内毒素的累积、分配、转化和排出等代谢过程影响。链状裸甲藻(Gymnodinium catenatum)能够产生麻痹性贝毒，是一种典型的有毒甲藻，在全球多地引发中毒事件，我国东海海域也曾暴发链状裸甲藻导致的麻痹性贝毒中毒事件。大部分麻痹性贝毒属于水溶性毒素，但近年来在链状裸甲藻中发现了一类具有脂溶性特征的新型麻痹性贝毒同系物，并依其来源命名为GC毒素(GC toxins)。目前，对于我国近海链状裸甲藻产生GC毒素状况及其在双壳类体内的代谢动力学过程和机理的认识仍然存在不足：我国近海链状裸甲藻产毒状况如何？受哪些因素影响？GC毒素在贝体内将会发生怎样的代谢动力学过程？其机理如何？回答这些问题有助于进一步评估链状裸甲藻所产GC毒素可能导致的中毒风险。对此，本研究建立了包括GC毒素在内的麻痹性贝毒液相色谱-质谱联用分析方法，分析了我国东海海域链状裸甲藻的产毒状况及影响因子，并通过模拟实验探究了GC毒素在毛蚶(Scapharca subcrenata)和扇贝(Argopecten irradians)等双壳贝类体内的累积、分配、转化和排出等代谢动力学过程和机理。

应用液-质联用技术建立了麻痹性贝毒检测方法，可以实现对包括GC毒素在内大部分麻痹性贝毒的分析。建立的液-质联用方法检出限在1.05-9.63 ng/g之间，定量限在3.15-28.90 ng/g之间，回收率基本介于80%–120%之间，满足分析需求。研究发现，贝类样品中的部分毒素基质效应超过60%，在分析样品时应通过基质加标方法对结果进行校正。该方法可实现对藻类和贝类样品中GC毒素的定性和半定量检测。

应用建立的液-质联用方法，分析了一株分离自我国东海的链状裸甲藻(MEL11株)产毒状况，并探究了温度和营养盐对链状裸甲藻产毒的影响。研究发现，链状裸甲藻MEL11株主要产生N-磺酰氨甲酰基类毒素和GC毒素，其中GC毒素可占藻细胞内毒素总量的50%以上。温度介于20-26^oC时链状裸甲藻最大细胞密度均大于0.70×10⁴ cells/mL，细胞毒素内含量介于189–219 fmol/cell间；表明20-26^oC范围内链状裸甲藻(MEL11株)生长与产毒状况较为稳定。氮限制和磷限制条件下链状裸甲藻MEL11株最大细胞密度降至0.3×10⁴ cells/mL以下，生长受到抑制；但该条件下链状裸甲藻生长至平台期和衰退期时细胞内GC毒素占比由超过50%降低至30%。以铵为氮源(88.3 mmol/L)时藻细胞内毒素含量可高达1 100±150 fmol/cell。研究结果揭示了我国近海链状裸甲藻产毒状况及其潜在影响因子。

通过室内模拟实验，探究了暴露于链状裸甲藻中的毛蚶和扇贝体内麻痹性贝毒的累积、分配、转化和排出过程，重点分析了GC毒素的代谢动力学特征。实验表明，毛蚶对麻痹性贝毒的累积效率仅为18.5%，低于扇贝的90%，对毒素的排出速率也仅为每天0.021。贝体内的GC毒素可通过R4位点羟基苯甲酸根的水解反应，转化生成对应的脱氨甲酰基类毒素。扇贝中GC毒素占比最高仅有7.7%，而毛蚶中GC毒素最高可达30%左右，表明扇贝转化GC毒素的能力强于毛蚶。内脏团是扇贝体内毒素蓄积的主要器官，但扇贝摄入链状裸甲藻后闭壳肌内的毒素含量也较高，可占扇贝体内毒素总量的30%以上，毒性最高可以达到  1 780±720 µg STXeq/kg。推测GC毒素的双亲性特征加快了其在不同组织间的毒素分配过程，导致闭壳肌中毒素含量上升。实验揭示了GC毒素有可能通过改变毒素分配过程增加麻痹性贝毒中毒风险，但其过程和机理仍有待于深入研究。

根据模拟实验结果，结合毒素在不同贝组织中的体外孵育实验，进一步探究了贝体内GC毒素的转化过程与机理。研究结果表明，毛蚶和扇贝组织中的脱氨甲酰基类毒素仅来自GC毒素的水解，而不是N-磺酰氨甲酰基类毒素。GC毒素的水解属于酶促反应，扇贝消化腺中水解酶活力高于其他组织。催化GC毒素水解的酶分子量应在50 kDa以上，最适反应温度为15–30℃，最适pH约为7.0。蛋白质组学结果表明，扇贝中存在的两种硝基苄基酯酶可能参与了GC毒素的催化水解。此外，毛蚶体内的GC毒素R1位点发生了羟基还原反应，但该反应在扇贝中没有出现。由于GC毒素的毒性尚未明确，目前仍难以准确评估GC毒素的水解和还原转化对其毒性的影响。

综上，本研究重点针对链状裸甲藻产生的GC毒素，构建了麻痹性贝毒的液-质联用分析方法，通过研究揭示了东海海域链状裸甲藻的产毒状况，发现我国近海的链状裸甲藻能够产生高比例的GC毒素，产毒状况受到营养盐种类和浓度的强烈影响；发现暴露于链状裸甲藻的扇贝闭壳肌中含有高含量的麻痹性贝毒，表明GC毒素有可能通过改变毒素分配过程增加麻痹性贝毒中毒风险；确认了GC毒素在双壳类体内的羟基还原反应和酶促水解反应，揭示了酶促水解反应在不同贝类、不同组织间差异和可能的水解酶。相关研究增进了对我国近海链状裸甲藻产毒特征的认识，深化了对贝体内GC毒素代谢动力学过程及机理的认知，为防控链状裸甲藻导致的麻痹性贝毒中毒风险提供了科学依据。

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids mainly produced by marine dinoflagellates. Bivalves may accumulate PSTs through feeding on toxic dinoflagellates, thus leading to human intoxication and, as a result, posing severe threat to human health. On one hand, the toxin composition and concentration of toxin-producing dinoflagellates strongly affect the toxin load and toxicity of bivalves. On the other hand, the biokinetic processes in bivalves including accumulation, distribution, biotransformation, and elimination may determine the toxin burden as well. Gymnodinium catenatum is an important PST producer that have caused multiple intoxication incidents around the world. Human poisoning events caused by G. catenatum also occurred in the costal areas of the East China Sea. Most PSTs are hydrophilic toxins, yet a novel group of lipophilic PSTs were revealed in G. catenatum recently and named GC toxins by their source. Little is known on the GC toxin production feature of G. catenatum from the East China Sea as well as the biokinetics of GC toxins in bivalves. What are the toxin production features of G. catenatum in the costal waters of China? How do environmental factors affect toxin production? What are the biokinetic processes of GC toxins in bivalves? What are the mechanisms behind such metabolic processes? Anwsering these questions helps assessing the risks of GC toxins produced by G. catenatum. The current dissertation estabilished an HPLC-MS method that is able to analyze PSTs including GC toxins, and examined the toxin production features of G. catenatum isolated from the East China Sea along with effects of environmental factors on toxin production feature. The biokinetic processes including accumulation, distribution, biotransformation, and elimination and the mechanisms behind these processes in blood clam (Scarpharca subcrenata) and bay scallop (Argopecten irradians) were revealed through simulation experiments.

In this study, an HPLC-MS method has been established, which could analyze most PST analogues including GC toxins. The limits of detection of PSTs range between 1.05–9.63 ng/g with the limits of quantitation between 3.15–28.90 ng/g. The recovery of most hydrophilic PSTs are between 80%–120%, which matchs the analytical requirements. The matrix effects of some PSTs in shellfish samples could exceed 60%, and the matrix-mixed standards should be used to reduce the strong matrix effect. The HPLC-MS method could be used to perform qualitative and semi-quantitative detection of GC toxins.

The toxin production features of G. catenatum (strain MEL11) isolated from the East China Sea were examined using the established HPLC-MS method, and the effects of temperature and nutrients on toxin production of G. catenatum was examined G. catenatum strain MEL11 mainly produce N-sulfocarbamoyl toxins and GC toxins that counts up to 50% of total PSTs in the algal cell. The maximum cell densities of G. catenatum (strain MEL11) cultivated in 20–26℃ exceeded 0.70×10⁴ cells/mL, and the cellular toxin concentration ranged between 189–219 fmol/cell, indicationg the growth and toxin production of were rather constant under 20-26^oC. When cultivated under nitrogen or phosphorus limited condition, the growth of G. catenatum were suppressed, maximum cell densities declined to 0.3×10⁴ cells/mL. Under nitrogen or phosphorus limiting condition, the proportion of GC toxins dropped from over 50% to around 30% when the culture reached stationary growth phase and decline phase. Cellular toxin concentration raised up to 1100±150 fmol/cell when replacing nitrate with ammonium at a concentration of 88.3 mmol/L. The results revealed the toxin production feature of G. catenatum in costal waters of China and the effects of temperature and nutrients.

The biokinetics including accumulation, distribution, biotransformation, and elimination of PSTs produced by G. catenatum in blood clam and bay scallop were examined by indoor simulation experiments focusing on the biokinetics of GC toxins. Toxin accumulation rate of blood clam was 18.5%, lower than of the bay scallop (90%). The detoxificaiton rate of blood clam was only 0.021 per day. The GC toxins were converted to decarbamoyl toxins through the hydrolysis of hydroxybenzoate group. The proportion of GC toxins remained under 7.7% in bay scallops, much lower than 30% in blood clams, suggesting that biotransformation of GC toxins were stronger in bay scallops. Most toxins accumulated in the viscera of bay scallop, yet the toxicity of adductor muscle in scallops exposed to G. catenatum reached 1780±720 µg STXeq/kg, with the total toxin burden of adductor muscle counts up to 30% of the whole scallop. The amphiphilicity of GC toxins probably accelerated its transfer between scallop tissues, which may lead to the high toxin concentration of adductor muscle. This discovery revealed that GC toxins may increase the risk of paralytic shellfish poisoning by prompting toxin distribution in scallops. The mechanisms, however, remain to be further investigated.

Based on the results of the simulating experiments, toxin biotransformation processes of GC toxins and its mechanism were further examined by in vitro incubation experiments. It was found that decarbamoyl toxins in the tissue homogenates of blood clam and bay scallop mainly derived from the hydrolysis of GC toxins, not N-sulfocarbamoyl toxins. The hydrolysis of GC toxins requires enzyme catalysis, the enzymatic activity governing the hydrolysis of GC toxins were higher in viscera of bay scallop than other tissues. The molecular weight of “GC toxin hydrolase” is likely larger than 50 kDa, with optimum temperature at 15–30℃ and optimum pH around 7.0. Two para-nitrobenzyl esterase-like proteins probably catalyse the hydrolysis of GC toxins. Besides, the reduction of hydroxyl on R1 of GC toxins were detected in blood clam, not bay scallops. Due to the limited knowledge on toxicity of GC toxins, the effects of hydrolysis and reduction of GC toxins on the toxicity of bivalves still remain to be further confirmed.

In general, this dissertation established an HPLC-MS method focusing on the analysis of GC toxins produced by G. catenatum. With the method, the toxin production features of a G. catenatum isolate from the East China Sea was revealed. The strain produces a high proportion of GC toxins, and its toxin profile are strongly affected by nutrient concentration and species of nitrogen. It was discovered, for the first time, that toxin content of the adductor muscle in bay scallop was high when scallops are exposed to G. catenatum. The results suggest that GC toxins might alter the distribution of PSTs and increase the risks of paralytic shellfish poisoning. Transformation of GC toxins, either through reduction of hydroxyl on R1 or enzymatic hydrolysis of hydroxybenzoate group on R4, were confirmed in bivalves. The potential enzymes involved in the hydrolysis of GC toxins were tentatively identified, and their activity in different species and tissues of bivalves were identified. The current study improved the understanding of toxin production of G. catenatum in the Chinese costal waters, gained further in-depth knowledge of GC toxin biokinetics in bivalves and its mechanism, and provided scientific basis of preventing the risks of PSTs caused by G. catenatum.

第1章 绪论... 1

1.1 麻痹性贝毒... 2

1.1.1 麻痹性贝毒的基本性质... 2

1.1.2 麻痹性贝毒的检测方法... 7

1.2 链状裸甲藻中的麻痹性贝毒及其危害... 10

1.2.1 链状裸甲藻... 10

1.2.2 链状裸甲藻产毒状况... 11

1.2.3 链状裸甲藻赤潮及其危害... 11

1.3 麻痹性贝毒在贝类体内的代谢... 13

1.3.1 麻痹性贝毒在贝类体内的累积、分配与排出... 13

1.3.2 麻痹性贝毒在双壳贝类中的转化... 15

1.3.3 麻痹性贝毒在贝类体内的代谢动力学研究... 17

1.3.4 GC毒素在贝类体内的代谢转化... 18

1.4 本研究的科学问题、目的和意义... 18

第2章 链状裸甲藻毒素液-质联用分析方法研究... 21

2.1 引言... 21

2.2 材料与方法... 21

2.2.1 仪器与试剂... 21

2.2.2 麻痹性贝毒提取与基质净化... 22

2.2.3 麻痹性贝毒分析方法的建立... 22

2.2.4 检测方法有效性评估... 25

2.3 结果... 27

2.3.1 麻痹性贝毒液-质联用分析方法... 27

2.3.2 分析方法评估... 29

2.4 讨论... 32

2.5 小结... 33

第3章 我国东海海域链状裸甲藻的产毒状况... 35

3.1 引言... 35

3.2 材料与方法... 36

3.2.1 试剂与仪器... 36

3.2.2 藻株分离和鉴定... 36

3.2.3 链状裸甲藻产毒特征及其受环境因子的影响实验... 36

3.3 结果... 38

3.3.1 链状裸甲藻的鉴定... 38

3.3.2 链状裸甲藻的产毒状况... 38

3.3.3 不同环境因子对链状裸甲藻生长与产毒状况的影响... 39

3.4 讨论... 41

3.4.1 GC毒素组成的多样性... 42

3.4.2 温度对链状裸甲藻生长和产毒状况的影响... 43

3.4.3 营养盐对链状裸甲藻生长和产毒状况的影响... 44

3.5 小结... 45

第4章 链状裸甲藻所产麻痹性贝毒在双壳类中的代谢动力学... 47

4.1 引言... 47

4.2 材料与方法... 48

4.2.1 试剂与仪器... 48

4.2.2 实验生物... 48

4.2.3 毛蚶染毒实验... 48

4.2.4 实验III. 49

4.2.5 扇贝染毒实验... 49

4.2.6 麻痹性贝毒分析... 50

4.2.7 数据分析... 51

4.3 结果... 52

4.3.1 产毒藻中麻痹性贝毒组成与含量... 54

4.3.2 毛蚶体内麻痹性贝毒的累积、转化和排出... 56

4.3.3 扇贝体内麻痹性贝毒的累积、分配、转化和排出... 61

4.4 讨论... 69

4.4.1 不同培养批次链状裸甲藻产毒状况差异... 70

4.4.2 麻痹性贝毒在毛蚶和扇贝体内的累积... 70

4.4.3 麻痹性贝毒在毛蚶和扇贝体内的转化... 72

4.4.4 麻痹性贝毒在毛蚶和扇贝体内的排出... 73

4.4.5 麻痹性贝毒在组织间的分配情况... 73

4.5 小结... 74

第5章 链状裸甲藻毒素在双壳类中的转化过程与机理... 77

5.1 引言... 77

5.2 材料与方法... 77

5.2.1 试剂与仪器... 77

5.2.2 实验生物... 77

5.2.3 毛蚶与扇贝组织体外毒素转化实验... 78

5.2.4 GC毒素水解酶活性测试... 81

5.2.5 利用蛋白质组学手段初步筛选GC毒素水解酶... 87

5.3 结果... 88

5.3.1 GC毒素在贝组织中的转化... 88

5.3.2 GC毒素水解酶活性测试... 92

5.4 讨论... 102

5.4.1 GC毒素的水解转化过程与机理... 102

5.4.2 贝类对GC毒素水解转化的种间与组织间差异... 103

5.4.3 GC毒素的其他转化反应... 105

5.5 小结... 106

第6章 结论、创新性与展望... 107

6.1 结论... 107

6.2 创新性... 108

6.3 展望... 108

参考文献... 111

附录一  缩略词表... 127

附录二  各类麻痹性贝毒名称... 129

致  谢... 131

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 135