细胞色素P450在拟柱孢藻毒素诱导小鼠肝毒性中的作用及机制研究

Cylindrospermopsin (CYN) is a toxic secondary metabolite from cyanobacteria, which poses a great threat to ecological security and human health. Liver has been shown to be the main target organ of CYN. The hepatic cytochrome P450 (CYP450) superfamily is a large group of monooxygenases metabolizing xenobiotics and endogenous substrates. It has been reported that CYP450 inhibitors could attenuate the toxicity of CYN, suggesting the involvement of CYP450 in the toxicity process of CYN. However, the mechanism of CYP450 in CYN-induced hepatotoxicity has not been revealed yet. In preliminary studies, we found that CYN-producing cyanobacteria showed a tendency of spreading from the south to the north in China. Furthermore, the concentration of CYN accumulated in some water bodies has exceeded a safety threshold. Under the worsening ecological situation globally, identifying molecular targets and elucidating toxicity mechanism of CYN are burning issues to be addressed. Therefore, we plan to use mice as toxicological models, and adopt various approaches such as RNA interference, histopathology, RNA-seq, iTRAQ quantitative proteomics, and recombinant protein, to achieve the following specific aims: ① To identify specific CYP450 members involved in the toxicity of CYN; ② To illuminate the mechanism of CYP450 in CYN-induced hepatotoxicity; and ③ To parse kinetic parameters of the interaction between CYN and CYP450. We anticipate identifying sensitive biomarkers, constructing a molecular response network of CYN in hepatotoxicity, and further defining correct targets for prevention and treatment of CYN-induced hepatotoxicity.

蓝藻次级代谢产物拟柱孢藻毒素（CYN）威胁到生态安全和人类健康，肝脏为其主要靶器官。研究表明肝脏中的氧化酶超家族细胞色素P450（CYP450）的抑制剂能缓解CYN的毒性，显示CYP450可能参与CYN的致毒过程，然而其作用机制尚不明晰。申请人前期研究发现我国产CYN的蓝藻呈现由南部向北部蔓延的趋势且部分水体的CYN浓度已超出安全阈值，因此鉴定CYN的作用靶点并阐明其致毒机制是当前毒理生态学中亟待解决的问题。本项目拟在前期工作基础上，通过建立小鼠毒理学模型，运用RNA干扰、组织病理、RNA-seq、iTRAQ定量蛋白质组学、重组蛋白等方法，开展以下研究：①鉴定介导CYN致毒的CYP450家族成员；②阐明CYP450在CYN诱导肝毒性中的作用机制；③解析CYN与CYP450相互作用的动力学特征。以期获得CYN诱导肝毒性的敏感生物标志物并构建分子应答网络，为CYN的危害防治提供重要靶点。

蓝藻次级代谢产物拟柱孢藻毒素（CYN）威胁到生态安全和人类健康，肝脏为其主要靶器官。我们前期发现我国产CYN的主要蓝藻在水体广泛存在并呈由南向北扩散之势，且部分水体的CYN浓度已超出安全阈值。本项目进一步分析了全球CYN污染情况，在欧洲、亚洲、大洋洲、北美和南美洲、以及非洲的共164个水体中，CYN浓度≥安全指导值1 μg/L的水体在各大洲占比为39.4%~75%，这种全球盛行的严峻态势凸显出CYN毒理学研究的急迫性。因此，我们通过腹腔单次注射CYN构建小鼠模型，探讨其发挥肝毒性的作用机制。主要结果如下：1）确定了CYN的半数致死剂量为150 µg/kg，为未来的体内毒理研究提供了剂量参考；2）获得了0、50、100、150和200 µg/kg CYN暴露下肝脏各类病理图片，揭示了CYN诱导的肝损伤包括肝小叶结构紊乱、肝细胞脂肪病变、空泡变性、坏死、炎症和纤维化，弥补了目前直观病理图片的稀缺；3）明确了CYN急性暴露主要导致脂肪病变，脂肪肝面积可超60%，表明CYN与非酒精性脂肪肝有关；4）转录组和蛋白组测序揭示了CYN急性暴露导致脂代谢紊乱，相关通路涉及非酒精性脂肪肝疾病、胆固醇代谢、脂肪酸合成与β氧化、脂质运输、类固醇合成、脂肪消化吸收等；5）鉴定了参与CYN肝毒性效应的8个CYP450（Cyp2c29、Cyp2c50、Cyp2c70、Cyp2e1、Cyp2f2、Cyp3a11、Cyp3a25、Cyp17a1），它们在介导外源物质代谢、脂类代谢和化学致癌中发挥重要作用；6）筛选了CYN分子应答网络中关联脂代谢的5个重要分子靶点（Apoa1、Apoa2、Apoc4、Apoe、Apoh）。本项目从全球格局评估CYN的暴露风险，将为CYN污染的风险管理和饮用水源地的选择提供科学指导。从组织病理和分子水平揭示CYN急性暴露诱导非酒精性脂肪肝，为蓝藻毒素与健康危害增添了新的证据。