虾夷扇贝毒素（yessotoxin）诱导的栉孔扇贝（Chlamys farreri）体内P糖蛋白转运活性及其分布研究

摘要

Many studies have shown that multi￣xenobiotic resistance (MXR) mediated by P￣glycoprotein (P￣gp) in shellfish played an important role in resisting or tolerating xenobiotics in polluted water. However, few research has been conducted whether this mechanism also works on the resistance or tolerance of dietary toxins (eg. yessotoxin, YTX) in shellfish. In this study, Chlamys farreri was exposed to the YTX toxin￣producing strain of Protoceratium reticulatum. YTX concentrations, P￣gp transport activity and P￣gp expression were measured and analyzed to study the changes over exposure time in gills and digestive gland of C. farreri. Consequently, it was discussed about the possible role of P￣gp in YTX toxin tolerance in shellfish. The results showed that YTX contents in gills and diges￣tive gland increased continuously with exposure time. Evident toxic effects were observed in the scallop tissues de￣spite no death resulted. After 12 h exposure, H.E. dye assay showed the melanization in the laterofrontal cillia of gill filament, exfoliation of epithelial cells in digestive gland and degradation of digestive ducts. Rhodamine￣B (RhoB) excretion experiments confirmed that YTX toxin, with a short exposure period, could significantly enhance the transport activity of P￣gp in the scallop. After 12 h exposure, the efflux rates of RhoB in gills and digestive gland were 3.8 and 1.4 times of those in unexposed ones;while later, the P￣gp activity had limited increase, and be￣gan to decrease after 72 h￣exposure. Contrary to the previous research, the inhibitor of P￣gp￣verapamil (VRP), in the given concentration, didn’ t inhibit but enhance the efflux of RhoB. Immunohistochemical analysis revealed in￣creased P￣gp expression in the laterofrontal ciliated columnar cells of gills, in the ductal epithelial columnar cells and digestive cells of digestive gland after YTX toxin exposure. Above all, this study suggested that YTX, possibly the substrate of P￣gp, can induce the transport activity and expression of P￣gp in C.farreri, and P￣gp played an im￣portant role in the YTX toxin tolerance. In addition, further studies were needed considering the discrepant results of VRP inhibiting the P￣gp activity, which was probably due to the different testing organisms used in this study.%大量研究表明P￣糖蛋白(P￣glycoprotein, P￣gp)介导的多型异源物质抗性机制(multi￣xentiobiotic resistance, MXR)在贝类耐受或抵抗污染水域中有毒有害物质的毒害中具有重要作用,但是该机制在贝类抵抗或耐受食源性毒素方面作用的研究却很少。本研究以栉孔扇贝(Chlamys farreri)为实验动物,向其投喂产虾夷扇贝毒素(yessotoxins, YTXs)的网状原角藻(Protoceratium reticulatum),通过检测栉孔扇贝鳃和消化腺组织中YTX毒素含量随时间的变化情况,结合2种组织中P￣gp介导的MXR活性变化及P￣gp在组织中的表达和分布,探讨P￣gp在扇贝耐受YTX毒素中可能的作用。研究结果表明,随着毒藻暴露时间的延长,栉孔扇贝鳃和消化腺中YTX含量逐渐增加,且具有明显的时间依赖关系。 YTX的大量累积虽未导致扇贝死亡,但是仍然对扇贝组织产生不同程度的毒性。苏木精￣伊红(hematoxylin￣eosin, HE)染色发现,染毒12 h后,栉孔扇贝鳃组织前端出现明显黑化,消化腺组织导管上皮细胞部分脱落入管腔中,腺管内细胞界限模糊,部分腺管崩解。罗丹明B(rhodamine B, RhoB)外排实验表明YTX毒素暴露能显著增强栉孔扇贝鳃和消化腺组织中P￣gp的转运活性,暴露12 h后鳃和消化腺RhoB的排出速率就已分别达到对照组的3.8倍和1.4倍,随着暴露时间的延长,P￣gp活性略有增强,72 h后开始下降；P￣gp的功能性抑制剂维拉帕米(verapamil, VRP)在实验设定的浓度下没有抑制反而增强了RhoB的外排；免疫定位显示,毒素暴露后栉孔扇贝鳃丝前端纤毛柱状细胞及消化腺导管上皮柱状细胞和腺管消化细胞P￣gp免疫阳性信号增强,说明YTX毒素暴露提高了P￣gp的表达量。综上推断,YTX毒素可能是P￣gp的底物,它能够诱导P￣gp转运活性增强,并增加P￣gp的表达量来参与扇贝对YTX毒素的耐受或抗性；设定浓度的VRP不能抑制P￣gp的活性,这可能是由于种属差异造成的,关于VRP对栉孔扇贝P￣gp的作用还有待进一步的研究。