基于质谱的代谢组学方法研究溴敌隆血液毒性及其作用机制

Bromadiolone poisoning is difficult to diagnose with a long latent period. The mechanisms of bromadiolone-induced hematotoxicity have not been fully elucidated, and may be associated with vitamin K cycle. As an indispensable technique in the context of molecular biology, metabonomics has become one of the most promising research tools in forensic toxicology. This study assessed the bromadiolone-induced hematotoxicity and its mechanisms using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), capturing bromadiolone-induced metabolic alterations using metabonomic analysis of plasma and urine. The metabolic differences between dosed group and control group were analysed using commercial software which revealed patterns in the multi-parametric dataset by principal component analyses (PCA) or partial least squares discriminant analysis (PLS-DA),to show the dose and time dependence of hematotoxicity of bromadiolone. The changes of endogenous metabolites associated with metabolic pathway were used to clarify the molecular mechanism of bromadiolone induced-hematotoxicity. Potential biomarker screening was performed according to VIP value, significant test and z-score. Mass spectrometry based-metabonomics was used to monitor and describe the changes in metabolic phenotypes at different stages of bromadiolone poisoning, providing biological data for the research of biomarkers.

溴敌隆中毒潜伏期长、极具隐匿性，其血液毒性确切作用机制尚未阐明，可能与维生素K循环有关。分子生物学研究发现代谢组学是法医毒理学最具应用前景的研究方法之一。本课题采用基于气相色谱-质谱联用和液相色谱-质谱联用的代谢组学研究方法，考察溴敌隆干预前后血浆和尿液代谢物谱的变化情况，对溴敌隆的血液毒性及其作用机制进行研究。采用主成分分析法(PCA)或偏最小二乘判别分析法(PLS-DA)分别研究对照组与溴敌隆给药组之间代谢谱差异，揭示溴敌隆血液毒性的时间-效应和剂量-效应规律；将内源性代谢产物的变化与其形成、转移机制和过程以及代谢途径相联系，从分子水平解释溴敌隆血液毒性作用机制，阐明其毒性效应；根据模型的变量重要因子(VIP值)、非参数检验结果和z值筛选潜在生物标志物。本课题利用基于质谱的代谢组学方法描述溴敌隆血液毒性发生、发展和恢复阶段机体代谢表型的改变，为生物标志物的动态跟踪鉴定打下基础。

溴敌隆是目前应用最为广泛的灭鼠药，其中毒潜伏期长、极具隐匿性，其血液毒性确切作用机制尚未阐明。本课题采用基于气相色谱-质谱联用和液相色谱-串联质谱的代谢组学研究方法，考察溴敌隆中毒后血浆和尿液代谢物谱的变化情况，采用主成分分析法(PCA)、偏最小二乘判别分析法(PLS-DA)、正交偏最小二乘法-判别分析(OPLS-DA) 对样品进行分型，建立可靠的预测模型，溴敌隆中毒大鼠和溴敌隆中毒患者组与正常对照组之间代谢组差异，发现溴敌隆血液毒性随着剂量增加而增强并存在时间依赖的特点。根据建立模型的变量重要因子(VIP值)和t检验结果筛选出潜在生物标志物，发现溴敌隆中毒引起酰基肉碱和胆酸血浆浓度的明显下降，C12至C18脂肪酸含量明显下降，但C7至C11脂肪酸含量明显升高，这两组脂肪酸最大的差别是长链脂肪酸转运进入线粒体基质需要肉毒碱酰化，短链脂肪酸则不需经过这一结合过程。除影响脂质代谢外，溴敌隆可能通过影响能量代谢、氨基酸代谢等多种代谢通路发挥其毒性作用。研究获得溴敌隆中毒机体改变和损伤的代谢层面的信息，为溴敌隆中毒法医学鉴定和临床诊断提供可行的技术支持。