Plakas Steven M, Wang Zhihong, El Said Kathleen R, Jester Edward L E, Granade Hudson R, Flewelling Leanne, Scott Paula, Dickey Robert W
Gulf Coast Seafood Laboratory, US Food and Drug Administration, P.O. Box 158, 1 Iberville Drive, Dauphin Island, AL 36528-0158, USA.
Toxicon. 2004 Nov;44(6):677-85. doi: 10.1016/j.toxicon.2004.07.027.
The metabolism and elimination of brevetoxins were examined in the Eastern oyster (Crassostrea virginica) following controlled exposures to Karenia brevis cultures in the laboratory. After a 2-day exposure period ( approximately 62 million cells/oyster), elimination of brevetoxins and their metabolites was monitored by using liquid chromatography/mass spectrometry (LC/MS). Composite toxin in oyster extracts was measured by in vitro assay (i.e. cytotoxicity, receptor binding, and ELISA). Of the parent algal toxins, PbTx-1 and PbTx-2 were not detectable by LC/MS in K. brevis-exposed oysters. PbTx-3 and PbTx-9, which are accumulated directly from K. brevis and through metabolic reduction of PbTx-2 in the oyster, were at levels initially (after exposure) of 0.74 and 0.49 microg equiv./g, respectively, and were eliminated largely within 2 weeks after dosing. PbTx-7 and PbTx-10, the reduced forms of PbTx-1, were non-detectable. Conjugative brevetoxin metabolites identified previously in field-exposed oysters were confirmed in the laboratory-exposed oysters. Cysteine conjugates of PbTx-1 and PbTx-2, and their sulfoxides, were in the highest abundance, as apparent in LC/MS ion traces, and were detectable for up to 6 months after dosing. Composite toxin measurements by in vitro assay also reflected persistence (up to 6 months) of brevetoxin residues in the oyster. Levels of cysteine conjugates, as determined by LC/MS, were well correlated with those of composite toxin, as measured by ELISA, throughout depuration. Composite toxin levels by cytotoxicity assay were well correlated with those by receptor binding assay. Cysteine-PbTx conjugates are useful LC/MS determinants of brevetoxin exposure and potential markers for composite toxin in the Eastern oyster.
在实验室中，将美国东牡蛎（Crassostrea virginica）置于短裸甲藻（Karenia brevis）培养物的受控暴露环境下，对短裸甲藻毒素的代谢和消除情况进行了研究。在为期2天的暴露期（约6200万个细胞/牡蛎）后，通过液相色谱/质谱联用仪（LC/MS）监测短裸甲藻毒素及其代谢产物的消除情况。通过体外检测（即细胞毒性、受体结合和酶联免疫吸附测定）来测量牡蛎提取物中的复合毒素。在暴露于短裸甲藻的牡蛎中，通过LC/MS无法检测到母体藻类毒素PbTx-1和PbTx-2。PbTx-3和PbTx-9可直接从短裸甲藻中积累，并通过牡蛎体内PbTx-2的代谢还原产生，最初（暴露后）的含量分别为0.74和0.49微克当量/克，并且在给药后2周内大部分被消除。无法检测到PbTx-1的还原形式PbTx-7和PbTx-10。在实验室暴露的牡蛎中证实了先前在野外暴露的牡蛎中鉴定出的结合型短裸甲藻毒素代谢产物。PbTx-1和PbTx-2的半胱氨酸结合物及其亚砜在LC/MS离子迹线中最为丰富，并且在给药后长达6个月内均可检测到。通过体外检测进行的复合毒素测量也反映了牡蛎中短裸甲藻毒素残留的持久性（长达6个月）。在整个净化过程中，通过LC/MS测定的半胱氨酸结合物水平与通过酶联免疫吸附测定测量的复合毒素水平密切相关。细胞毒性检测的复合毒素水平与受体结合检测的水平密切相关。半胱氨酸-PbTx结合物是短裸甲藻毒素暴露的有用LC/MS测定指标，也是美国东牡蛎中复合毒素的潜在标志物。
