双壳贝类细胞色素P450及其在腹泻性贝毒代谢解毒机制中的作用研究

Diarrhetic shellfish poisoning (DSP) toxins, one of the most important shellfish toxins, are found in the sea area all over the world. DSP toxins can lead to diarrhetic poisoning of humanity, and moreover, induce tumor. But bivalves have some tolerance to DSP toxins, and various bivalve species exhibit different sensibility to the toxicity of DSP toxins. Nevertheless, the metabolism and detoxification of DSP toxins in bivalve species remains unclear. Cytochrome P450 (CYP450) enzymes is a very important superfamily involved in detoxification and resistance, which mainly transform liposoluble toxic chemicals into hydrosoluble substances easily eliminated. In this study, in vitro metabolism experiment and integral animal experiment will be conducted to explore the roles of CYP450 in metabolism and detoxification of DSP toxins in bivalve. RNA sequencing and de novo assembly of the digestive gland and gill transcriptome in Perna viridis fed with toxic Prorocentrum lima will be firstly done, and expression profiles of CYP450 isoforms involved in detoxification of DSP toxins will be analyzed. Then, metabolism of okadaic acid (OA), a main component of DSP toxins will be performed in vitro, and metabolites of OA will be analyzed. Thirdly, some potential CYP450 isoforms involved in DSP toxin detoxification in bivalve will be silenced by RNAi technique, and effect of silencing genes on toxins metabolism and susceptibility of bivalve to OA will be investigated to distinguish which isozymes are responsible for DSP toxin detoxification. Finally, the responses, DSP content and CYP450 isoforms expression will be compared between different bivalve species after exposed to P. lima. Our project will provide new insights into the metabolism and detoxification of DSP toxins in bivalve species.

腹泻性贝毒是最常见的贝毒之一，人们误食其污染的贝类时，可造成腹泻性中毒，甚至可引致肿瘤发生。但贝类对其有一定的耐受性，且不同贝类对毒素的毒性响应存在很大差异。我们推测，生物转化中重要的I相酶细胞色素P450（CYP450）可能在DSP毒素的代谢解毒过程中发挥重要作用。本课题采取体外代谢实验和贝类整体动物实验相结合的手段，以翡翠贻贝等为实验对象，通过染毒后贝类的转录组研究，DSP毒素主要成分OA的体外代谢分析，细胞色素P450不同亚型基因的表达与毒素代谢的关系分析，以及不同贝类细胞色素P450亚型基因表达与毒素积累和对毒素毒性响应的比较分析等，研究毒素的代谢途径，探讨细胞色素P450在DSP毒素代谢解毒中的作用，为深入认识DSP毒素在贝体中的代谢解毒过程和耐受机制，明确不同贝类对毒素相应差异的原因奠定基础，为染毒贝类的净化与贝类的健康养殖提供理论基础。

以利玛原甲藻（Prorocentrum lima）为DSP毒素的来源，通过转录组测序和qPCR分析了DSP毒素对翡翠贻贝（Perna viridis）消化腺中重要代谢酶系基因表达的影响。结果显示，暴露6h时，解毒和代谢相关基因如CYP3A-like、ABCB10、ABCC1、ABCG-like、GST-σ3显著上调；暴露4day后，IAP、Nrf2及其下游基因GST-σ3、ABCG-like、ABCB10上调，而Keap1表达下调。此外，染毒后，一些与细胞骨架和免疫相关的基因表达也发生了变化。我们推测短时间染毒时，翡翠贻贝主要通过GST和ABC转运蛋白解毒，较长时间染毒会激活Nrf2信号途径和IAP，进而降低DSP毒素对贻贝的损伤。DSP毒素可引起贝类细胞骨架不稳定，并对其免疫系统产生影响。. 进一步研究发现，利玛原甲藻染毒可引起翡翠贻贝鳃组织和消化腺中CYP3A4、CYP2D14-like、CYP3L3和CYP2C8等重要CYP450亚型mRNA的表达变化。CYP3A4特异性抑制剂酮康唑可显著抑制翡翠贻贝消化腺组织中CYP3A4活性，并抑制OA在消化腺组织中的积累，提示CYP3A4活性的改变可改变毒素在贝体中的代谢过程，CYP3A4在DSP毒素代谢中发挥重要作用。采用液相色谱质谱发现，翡翠贻贝消化腺匀浆可将OA代谢成母离子为m/z 819，碎片离子为m/z为563和255的产物，证实翡翠贻贝消化腺对OA确实具有一定的转化能力。. 姜黄素、肉桂醛、槲皮素、冬凌草甲酸和大蒜素等5种天然活性物质对翡翠贻贝消化腺组织中DSP毒素积累的影响研究发现，姜黄素和肉桂醛可显著抑制DSP毒素在贻贝消化腺中的积累，并减轻其对消化腺的损伤。进一步分析发现，姜黄素和肉桂醛可显著抑制DSP毒素诱导的核受体RXR、AhR和CYP3A4的表达。我们推测，肉桂醛和姜黄素对DSP毒素积累的抑制可能与肉桂醛诱导的CYP3A4表达降低有关。研究结果为贝类毒素的净化提供了重要依据。. 另外，项目还使用脊椎动物模型鸡胚研究了OA对鸡胚血管发生、神经嵴细胞产生的影响，通过iTRAQ技术探究了抑食金球藻对贝类危害的原因及机理。