| Yessotoxins(YTXs) represent a group of lipophilic polycyclic ether compounds with two sulfate groups. They are mainly produced by marine dinoflagellate Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. By filter-feeding these algae, shellfish can accumulate and transfer yessotoxins along the food chain and pose a threat to human health.In this study, the influence of temperature and N/P ratios on the growth and toxin production of P. reticulatum, isolated from the Northern Yellow Sea of China recently, were investigated firstly. On this basis, Chlamys farreri was selected as test organism in the next research to accept a short-term exposure of P. reticulatum. The accumulation of YTX, the transport activity and expression of P-glycoprotein(P-gp) closely related to the multixenobiotic resistance in the gill and digestive galand of C. farreri were studied by HPLC-MS/MS analysis, rhodamine-B(Rho B) excretion experiments and immunohistochemical technique respectively. The mRNA expressions of P-gp and other three proteins associated with detoxification were also investigated over exposure time in gills and digestive gland of scallop. These three proteins are multidrug resistance-associated protein(MRP), cytochrome P450 3A-like proteins(CYP3A) and glutathione-S-transferase omega class(GST-ω). Furthermore, possible roles and machanisms of the four ptoreins in the resistance and detoxification of YTX in scallop were discussed. The main experimental results are as follows:(1)The effects of temperature and N/P ratios on the growth and toxin production of P.reticulatumResults indicated that lower temperature was more suitable for P. recticulatum. The maximum specific growth rate reaching to 0.34/d in the exponential growth phase was observed in the in L1-Si medium at 15℃ N- and P-limitation also led to the deformation and increase of cell size in the stationary phase respectively. These morphological changes had nothing to do with temperature. HPLC–MS/MS analysis suggested that YTX was the main toxin products of P.reticulatum. Both N and P limitation were good for the toxin intracellular accumulation. The maximum concentration reached to 92.6pg/cell in 1/10 P medium at 15℃, which was 3.8 times and 7.1 times in 1/10 N medium and L1-Si medium respectively. Temperature had opposite influence on the toxin content under N- and P-limitation: YTX content per cell decreased with the temperature rise in the range of 5℃-15℃ under N-limitation, but the trend was opposite in the P-limitation medium.( 2) The transport activity and expression of P-glycoprotein in gills and digestive gland of Chlamys farreri induced by YTXRhodamine-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 hr 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 began to decrease after 72h-exposure. Contrary to the previous research, the inhibitor of P-gp, verapamil(VRP), in the given concentration, didn’t inhibit but enhanced the efflux of RhoB. Immunohistochemical analysis revealed increased 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.(3)The mRNA expressions of P-gp and other three proteins associated with detoxification in gills and digestive gland of Chlamys farreriWith a short exposure period, real-time fluorescent quantitative PCR indicated that gene expression of P-gp both in gills and digestive gland of C. farreri up-regulated remarkablely; the gene expressions of GST-ω and MRP in gills also amplified, increasing 8.3 and 4.5 times respectively; CYP3 A in gills and GST-ω and MRP in digestive gland increased but had no significant difference; CYP3 A in digestive gland was significantly inhibited by YTX and dropped to the half of the control. It suggested that all these genes were probably involved in the detoxification process with different roles. With increased exposure time, expression of these genes in gills and digestive gland were gradually decreased; expressions of MRP in gills and GST-ω and MRP in digestive gland were even prominently inhibited after 72h-exposure. It may be related to accumulation of YTX and tissue damages.Above all, the results of this study suggested that both temperature and N/P ratio had influence on growth and toxin production of this P.reticulatum strain, and more significant for N/P ratio. L1-Si medium at 15℃ and 1/10 P medium at 15℃ was best for the growth and the YTX production of P.reticulatum respectively. YTX, the possible substrate of P-gp, could induce the transport activity, concentration and mRNA expression of P-gp in C. farreri. Therefore, P-gp was likely to play an important role in the YTX toxin tolerance and could be used as a biomarker candidate for indicating YTX content in water environment. Although metabolic enzymes CYP3 A and GST-ω and transport protein MRP in C. farreri also took part in the detoxification process, further studies were needed to explain their detailed functions in this process. |
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