Ishihara Kenji, Katsutani Naruo, Asai Naoki, Inomata Akira, Uemura Yuji, Suganuma Akiyoshi, Sawada Kohei, Yokoi Tsuyoshi, Aoki Toyohiko
Tsukuba Research, Drug Safety Research Laboratories, Eisai Co, Ltd, Tsukuba, Japan.
Basic Clin Pharmacol Toxicol. 2009 Sep;105(3):156-66. doi: 10.1111/j.1742-7843.2009.00410.x. Epub 2009 Apr 8.
This (1)H nuclear magnetic resonance metabonomics study was aimed to determine urinary biomarkers of cholestasis resulting from inhibition of biliary secretion of bile or obstruction of bile flow. To inhibit biliary secretion of bile, cyclosporine A was administered to male Sprague-Dawley rats. Obstruction of bile flow was induced by administration of 4,4'-methylene dianiline, alpha-naphthylisothiocyanate or bile duct ligation. Clinical pathological and histopathological examinations were performed to confirm cholestatic injury and (1)H nuclear magnetic resonance spectral data for urine samples were analysed to determine similarities and differences in profiles of metabolites using the Spotfire. In cyclosporine A-treated groups, serum total bilirubin and bile acid were significantly increased but no remarkable hepatic histopathological-changes were observed. In 4,4'-methylene dianiline-, alpha-naphthylisothiocyanate- and bile duct ligation-treated groups, serum alkaline phosphatase, gamma-glutamyltranspeptidase and total bilirubin levels increased significantly, and hepatic histopathological-changes were observed. On urinary (1)H nuclear magnetic resonance spectral analysis, area intensities derived from 0.66 to 1.90 ppm were decreased by cyclosporine A, whereas they were increased by other treatments. These metabolites were identified using the NMR suite as bile acids, branched-chain amino acids, n-butyrate, propionate, methyl malonate and valerate. These metabolites were further investigated by K-means clustering analysis. The cluster of these metabolites is considered to be altered by cholestasis. We conclude that bile acids, valine and methyl malonate have a possibility to be urinary cholestatic biomarkers, which distinguish a difference in mechanism of toxicity. (1)H nuclear magnetic resonance metabonomics thus appears to be useful for determining the mechanisms of toxicity and can be front-loaded in drug safety evaluation and biomarker discovery.
这项氢核磁共振代谢组学研究旨在确定胆汁分泌受抑制或胆汁流动受阻导致胆汁淤积的尿液生物标志物。为抑制胆汁分泌，将环孢素A给予雄性Sprague-Dawley大鼠。通过给予4,4'-亚甲基二苯胺、α-萘异硫氰酸酯或胆管结扎诱导胆汁流动受阻。进行临床病理和组织病理学检查以确认胆汁淤积性损伤，并使用Spotfire分析尿液样本的氢核磁共振光谱数据，以确定代谢物谱的异同。在环孢素A处理组中，血清总胆红素和胆汁酸显著升高，但未观察到明显的肝脏组织病理学变化。在4,4'-亚甲基二苯胺、α-萘异硫氰酸酯和胆管结扎处理组中，血清碱性磷酸酶、γ-谷氨酰转肽酶和总胆红素水平显著升高，并观察到肝脏组织病理学变化。对尿液氢核磁共振光谱分析显示，环孢素A使0.66至1.90 ppm处的面积强度降低，而其他处理使其升高。使用NMR套件将这些代谢物鉴定为胆汁酸、支链氨基酸、正丁酸、丙酸、甲基丙二酸和戊酸。通过K均值聚类分析对这些代谢物进行进一步研究。这些代谢物的聚类被认为因胆汁淤积而改变。我们得出结论，胆汁酸、缬氨酸和甲基丙二酸有可能成为区分毒性机制差异的尿液胆汁淤积生物标志物。因此，氢核磁共振代谢组学似乎有助于确定毒性机制，并可用于药物安全性评价和生物标志物发现的前期研究。
