吡咯里西啶生物碱-DNA加合物作为生物标志物的毒代动力学评价及加合位点研究

Liver diseases with unknown etiology (excluded virus, alcohol, etc. induced) have become an important part in the liver diseases of China. However, the patients were usually not being treated properly due to the lack of specific diagnostic methods, which thus resulted in the worse progression of disease. Pyrrolizidine alkaloids (PAs) are probably the most common phytotoxins that induce liver injury in humans and livestock. PAs are common constituents of hundreds of plant species around the world. It is reported that about 3% of the world’s flowering plants contain PAs. PAs-induced liver disease is mainly due to the consumption of the PAs-containing plants used for herbal medicines, herbal teas, vegetables, and dietary supplements, as well as unwitting consumption of PAs-contaminated foodstuffs, such as grain crops, honey, milk, and eggs, leading to acute liver injury and chronic liver diseases including liver cancer. However, the definitive diagnosis of liver diseases associated with PAs is highly challenging due to the lack of characteristic clinical features and specific diagnostic method. PAs are pro-toxins, which undergo metabolic activation to generate dehydropyrrolizidine alkaloids (pyrrolic esters). Pyrrolic ester are chemically and biologically reactive, capable of alkylating cellular macromolecules such as DNA to form pyrrole-DNA adducts, which are highly related to the tumorigenicity of PAs. Previous studies have demonstrated that pyrrole-DNA adducts could potentially to be a specific biomarkers for the diagnosis of PAs intoxication. However, their in vivo elimination rate as well as the relationship with the carcinogenic mechanism has never been investigated, which obstructed their further development to be a rational and mechanism-based biomarker. In the proposed study, the toxicokinetic of pyrrole-DNA adducts in mice will be investigated and evaluated; their adduction hotspots on the pro-oncogene K-ras will be accurately located and the relationship with mutation/cancer will be illustrated. The outcomes of proposed study are anticipated to provide scientific evidence for the development of pyrrole-DNA adducts to be a mechanism-based biomarker for PAs-intoxication. Besides, the definitive diagnosis of PAs-related liver disease was also considered to be helpful for the diagnosis of liver diseases with unknown etiology.

不明原因肝病已成为我国肝病的重要组成部分。因其缺乏特异性诊断方法，病人不能及时得到诊治而使病情延误。吡咯里西啶生物碱（PAs）广泛分布于自然界，可引起肝窦阻塞综合征等毒性症状；还可通过间接食物链污染日常食用的蜂蜜、牛奶、谷物等，导致慢性肝病如肝癌的发生。因缺乏典型症状及特异性检测手段，很多PAs中毒病人往往被误诊、漏诊。前期研究表明PAs经体内代谢活化产生的吡咯-DNA加合物极有可能成为检测PAs中毒的特异性生物标志物。然而其在体内的消除速率及与致病机制的关系尚未阐明，直接影响其是否能成为合理可行、基于毒性机理的理想生物标志物。本项目拟对吡咯-DNA加合物在动物体内的毒代动力学进行评价；定位其在致癌关键基因序列上的加合位点，阐明其与突变/肿瘤之间的直接联系。本项目将为吡咯-DNA加合物发展成为表征PAs中毒的特异性生物标志物提供科学依据；也期为临床上确诊与PAs有关的不明原因肝病提供助力。

不明原因肝病已成为我国肝病的重要组成部分。因其缺乏特异性诊断方法，病人不能及时得到诊治而使病情延误。吡咯里西啶生物碱（PAs）广泛分布于自然界，可引起肝窦阻塞综合征等毒性症状；还可通过间接食物链污染日常食用的蜂蜜、牛奶、谷物等，导致慢性肝病如肝癌的发生。因缺乏典型症状及特异性检测手段，很多PAs中毒病人往往被误诊、漏诊。前期研究表明PAs经体内代谢活化产生的吡咯-DNA加合物极有可能成为检测PAs中毒的特异性生物标志物。然而其在体内的消除速率及与致病机制的关系尚未阐明，直接影响其是否能成为合理可行、基于毒性机理的理想生物标志物。通过对本项目的研究，我们阐明了吡咯─DNA加合物在动物体内生成的剂量依赖性；对其在单次给药及低剂量多次给药模型下的清除速率（Ke）和消除半衰期（t1/2）等毒代动力学规律进行了动态研究和评价；同时，我们还对低剂量长期摄入PAs的风险性进行了科学性评估。此外，吡咯─DNA加合物在核苷酸序列上的损伤位点以及与癌变突变的联系也得到了进一步的确证。考察及验证吡咯─DNA加合物在作为PAs中毒诊断生物标志物方面的可行性及机理相关性，不仅有助于建立PAs中毒的特异性诊断措施，而且有望为与PAs有关的不明原因的肝病诊断提供助力。