应用代谢组学分析2,3,7,8-四氯二苯并二恶英毒性与肝癌相关的代谢机理研究

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of persistent organic pollutants which is a notorious environmental toxicant. Previously, we carried out mass spectrometry-based metabolomics for measurement of the metabolites in tissues and blood of mice and found that succinate was elevated up to 11-fold in liver tissue of aryl hydrocarbon receptor (AhR)-sensitive mice exposed to TCDD compared to control group, indicating that succinate dehydrogenase was inhibited by TCDD exposure. Therefore, we will investigate the toxicity in normal liver cell line LO2 when exposed to TCDD. Isotopic labelled glucose or glutamine will be used in cell culture. Metabolic flux analysis will be conducted by using mass spectrometry. Warburg effect, which is the hallmark of cancer cells, as well as the expression changes of succinate dehydrogenase will also be observed. To confirm the metabolic regulation of succinate dehydrogenase, we will knock down the encoding gene of succinate dehydrogenase in LO2 cells and figure out the metabolic switch by metabolomics combining with isotopic labelling. Furthermore, we will treat TCDD to AhR-sensitive mice for understanding the toxic effects of TCDD. To further interrogate the contribution of TCDD to tumorigenesis in animal bodies, the bear-tumor mice will be exposed to TCDD for understanding the promotion of liver tumor by metabolomic approach based on isotopic labeling. In a word, metabolomics with isotopic labelling and molecular biology will be used to uncover the metabolic mechanisms associated with liver cancer development induced by TCDD exposure via inhibition of succinate dehydrogenase.

二恶英是具有促癌作用的持久性有机污染物。在前期工作中，基于质谱的代谢组学方法分析了动物组织与血液中的代谢轮廓受到2,3,7,8-四氯二苯并二恶英（TCDD）的扰动，发现芳香烃受体敏感型小鼠的肝脏中琥珀酸浓度升高达11倍之多，据此推测琥珀酸脱氢酶受到抑制，但其代谢机理至今未明。因此，本项目拟在TCDD暴露下的细胞培养中采用稳定同位素标记的葡萄糖或谷氨酰胺，运用质谱分析代谢流，观察瓦尔伯格效应（Warburg effect）是否发生。为了验证琥珀酸脱氢酶的代谢调控功能，还将沉默琥珀酸脱氢酶的编码基因并分析代谢流变化。最后在动物实验中，把微量的TCDD灌胃予荷瘤的小鼠判别TCDD是否可促进肝脏肿瘤的生长，静脉注射同位素标记的葡萄糖进行组织代谢分析，同时进行免疫组织化学检测。本项目预期可达到描述TCDD暴露抑制琥珀酸脱氢酶而改变代谢流的规律，进而解释TCDD促进肝癌发生发展的代谢机理。

项目背景：小分子代谢物最接近于表型，可反映生理病理变化。代谢组学作为系统生物学的下游，通过高通量质谱分析技术，可以定性定量数百至数千个代谢物。故检测小分子代谢物，特别是把稳定同位素标记的代谢流分析技术与分子生物学结合起来，有助于理解疾病发生发展的进程和代谢调控机制。本项目通过开发运用代谢组学技术，阐明肿瘤代谢相关的调控机制。.主要研究内容：针对2,3,7,8-四氯二苯并二恶英导致肝毒性的代谢组变化，肝癌发生发展，肝癌干细胞，常山酮治疗结直肠癌以及其他肿瘤代谢的机制开展了一系列研究工作。.重要结果：课题组开展了基于质谱的代谢组学与代谢流技术开发，进而把代谢组学与代谢流分析技术，跟细胞分子生物学结合起来阐明肿瘤代谢调控机制。首先，我们运用质谱技术发展了定量分析还原型和氧化型的辅酶Q10，在此基础上，进一步分析了暴露于2,3,7,8-四氯二苯并二恶英（TCDD）的小鼠肝脏组织，发现TCDD暴露可以显著抑制辅酶Q10的合成。其次，我们课题组还开展了多个肿瘤代谢相关的研究项目，包括：发现己糖胺合成通路是CD133阳性肿瘤干细胞维持干性的关键代谢通路；癌基因真核启动因子5A2（eIF-5A2）过表达可通过糖脂代谢促进肝癌的发生发展；在缺氧条件下肿瘤细胞可以摄取乙酸，促进关键基因的组蛋白乙酰化与癌细胞存活的表观遗传学机制；化合物常山酮通过下调Akt/mTORC1信号通路与糖酵解，抑制结肠癌生长；进一步还发现常山酮可以根据营养条件的改变而双向调控自噬，通过ULK1的不同磷酸化位点与自噬相关基因Atg7均可抑制结肠癌生长。再次，申请人还参与了多个合作课题，通过代谢组学与代谢流分析技术，确证了肝癌中转酮醇酶的代谢重编程机制；验证了磷酸甘油变位酶1在肿瘤细胞中调控核苷合成的机制。.科学意义：基于质谱的代谢组学与代谢流分析技术的开发与应用，极大地推动了代谢领域的发展。通过该技术的应用，使我们更进一步认识TCDD的毒性机制，肿瘤细胞新陈代谢的动态变化与调控机制，有助于理解肿瘤的代谢异常并鉴定其治疗靶点。