辛硫磷与鲫鱼肝CYP450作用机理研究及作用位点分析

Freshwater fish is widely favoured by Chinese people, its edible safety is an issue with highly significance. As the increasing of the amount of chemical pesticides utilized in freshwater fish feeding, their effect on the edible safety of freshwater fish attracted more and more attention nowadays. The lack of pesticides standards have led to intensive safety risks for aquatic products. This project employed the widespread carassius auratus gibelio as the symbol specie and phoxim as the target analyte, a series of fundamental research using carassius auratus gibelio, which was polluted by phoxim, was performed. This project involved the identification of CYP450 isoforms, evaluation of effect of phoxim on mRNA expression of CYP isoforms, the interactional sites were also studied by MALDI-TOF MS technique. The affect of phoxim on structural change of protein molecular was researched by modern analysis technology including AFM, CD, to explain the mechanism of interaction between phoxim and CYP450. The 3D-QSAR model of CYP450 acting on phoxim was established by means of computer simulate technology, which was aiming to study the structure-activity relationship between phoxim and CYP450, to predict the characteristic of the major metabolite,and to indicate its pathway and mechanism. The research results could offer the theoretical basis for establishing pesticide safety standard,controlling the harm of pesticide residues to ensure the edible safety of aquatic products.

淡水鱼类是深受我国人民喜爱的食品，其食用安全问题意义重大。而随着化学农药使用量的增加，其对淡水鱼类的安全影响日益引起关注。针对相应农药标准缺乏从而导致水产食品安全隐患的现状，本项目以有机磷农药辛硫磷为化学农药代表，以分布广泛的鲫鱼为淡水鱼类代表，开展系统的基础研究。包括农药代谢关键酶CYP450酶系的亚型鉴定，辛硫磷对CYP450亚型mRNA及蛋白表达的影响，并运用MALDI-TOF MS技术鉴定作用位点；运用AFM、CD等现代仪器技术研究辛硫磷对CYP450结构变化影响，阐释辛硫磷与CYP450作用机理。采用计算机模拟技术，构建辛硫磷与CYP450作用的3D-QSAR模型，推断辛硫磷作用位点及与CYP450酶之间的构效关系，预测辛硫磷代谢产物特征，揭示其作用途径和机理。为合理制定农药安全性标准，控制农药残留危害从而保障淡水产品食用安全奠定基础。

本项目研究了800字以内。.一、研究背景：. 开展有机磷农药在水生动物体内残留代谢的基础研究，明确其代谢产物并探讨其途径，进一步研究其机制从而进行控制，已成为合理制定安全用药标准及施药期和休药期、正确判断农药的特殊毒理、水产生态毒性，使得化学农药的监控有据可依，从而合理监控以降低外源性化学农药危害的前提；亦是保障水产品食用安全和人体健康，促进出口贸易发展的社会需求。 .二、主要研究内容及重要结果.研究内容.1.1辛硫磷对鲫鱼肝细胞色素P450不同亚型体内体外代谢活性的影响.1.2辛硫磷对鲫鱼肝CYP450亚型 mRNA表达调控的研究. 1.3辛硫磷与CYP450亚型蛋白质作用机理研究.1.4辛硫磷与CYP450作用的QSAR模型构建及作用位点的分析.关键数据.1. 六种CYP450同工酶CYP3A4、CYP1A2, CYP2D6, CYP2E1, CYP2C9 和 CYP2C19的IC50值分别为：6.096µmol/L、3.291µmol/L、12.25µmol/L、6.194µmol/L、7.427µmol/L、10.25µmol/L。.2. 结合上述Topomer CoMFA模型、CoMFA模型、CoMSIA模型以及对接结果进行综合分析，推测辛硫磷与CYP450酶作用方式主要是氢键作用。辛硫磷的乙氧基中的O原子与CYP450酶的氨基之间的氢键在有辛硫磷与CYP450酶相互作用中发挥了重要作用。具体的结合方式是辛硫磷中乙氧基中的O原子与CYP3A4亚型中的氨基酸ILE443的氢原子形成氢键。辛硫磷与CYP450结合后经过脱硫氧化、水解等反应生成辛氧磷、DETP等产物。.3. 辛硫磷对CYP1A活性产生抑制影响，辛硫磷对CYP3A酶活性整体上呈现较为明显的抑制作用。.4. 辛硫磷对鲫鱼肝脏CYP1AmRNA及CYP3AmRNA的表达整体上呈现较为明显的下调作用。.5. 辛硫磷对CYP1A蛋白表达量的影响整体上相较各对照组都呈现极显著的差异（P ＜ 0.01）；各时间组间不同浓度染毒组的蛋白表达量都呈现相同的剂量-效应影响；CYP1A蛋白表达量在不同浓度组间都呈现明显的时间-效应影响；辛硫磷会降低CYP3A蛋白表达量。.四、科学意义.项目预期研究成果可为有效控制化学农药在淡水鱼体内残留，从而保障淡水产品食用安全奠定理论基础；同时，也可为政府和管理部门的