Pearson P G, Omichinski J G, Holme J A, McClanahan R H, Brunborg G, Søderlund E J, Dybing E, Nelson S D
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle 98195.
Toxicol Appl Pharmacol. 1993 Feb;118(2):196-204. doi: 10.1006/taap.1993.1025.
Analogs of tris(2,3-dibromopropyl)phosphate (Tris-BP) either labeled at specific positions with carbon-14 and phosphorus-32 or dual-labeled with both deuterium and tritium were administered to male Wistar rats at a nephrotoxic dose of 360 mumol/kg. The covalent binding of Tris-BP metabolites to hepatic, renal, and testicular proteins was determined after 9 and 24 hr, and plasma concentrations of bis(2,3-dibromopropyl)-phosphate (Bis-BP) formed metabolically from Tris-BP were measured at intervals throughout the initial 9-hr postdosing period. The covalent binding of 14C-Tris-BP metabolites in the kidney (2495 +/- 404 pmol/mg protein) was greater than that in the liver (476 +/- 123 pmol/mg protein) or testes (94 +/- 11 pmol/mg protein); the extent of renal covalent protein binding of Tris-BP metabolites was decreased by 82 and 84% when deuterium was substituted at carbon-2 and carbon-3, respectively. Substitution of Tris-BP with deuterium at carbon-2 or carbon-3 also decreased the mean area under the curve for Bis-BP plasma concentration by 48 and 57%, respectively. The mechanism of Tris-BP-induced renal and hepatic DNA damage was evaluated in Wistar rats by an automated alkaline elution procedure after the administration of analogs of Tris-BP or Bis-BP labeled at specific positions with deuterium. Renal DNA damage was decreased when Tris-BP was substituted with deuterium at either carbon-2 or carbon-3; the magnitude of the change correlated with both a decrease in the area under the Bis-BP plasma curve and a decrease in renal covalent binding of Tris-BP metabolites for each of the deuterated analogs. In marked contrast, analogs of Bis-BP labeled with deuterium at carbon-2 or carbon-3 did not show a decrease in the severity of renal DNA damage compared to unlabeled Bis-BP. On the basis of these observations a metabolic scheme for hepatic P-450-mediated oxidation at either carbon-2 or carbon-3 of Tris-BP affording Bis-BP by two alternate pathways that are susceptible to primary deuterium kinetic isotope effects is proposed. The Tris-BP metabolite, Bis-BP, is subsequently metabolized to reactive intermediates that cause DNA damage and bind to kidney proteins in a mechanism independent of cytochrome P-450.
将标记有碳 - 14和磷 - 32的特定位置的磷酸三（2,3 - 二溴丙基）酯（Tris - BP）类似物或同时用氘和氚进行双标记的类似物，以360 μmol/kg的肾毒性剂量给予雄性Wistar大鼠。在9小时和24小时后测定Tris - BP代谢物与肝、肾和睾丸蛋白质的共价结合，并在给药后的最初9小时内每隔一段时间测量由Tris - BP代谢形成的双（2,3 - 二溴丙基）磷酸酯（Bis - BP）的血浆浓度。14C - Tris - BP代谢物在肾脏中的共价结合（2495±404 pmol/mg蛋白质）大于在肝脏（476±123 pmol/mg蛋白质）或睾丸（94±11 pmol/mg蛋白质）中的共价结合；当在碳 - 2和碳 - 3处分别用氘取代时，Tris - BP代谢物的肾脏共价蛋白质结合程度分别降低了82%和84%。在碳 - 2或碳 - 3处用氘取代Tris - BP也分别使Bis - BP血浆浓度曲线下的平均面积降低了48%和57%。在用在特定位置标记有氘的Tris - BP或Bis - BP类似物给药后，通过自动碱性洗脱程序在Wistar大鼠中评估Tris - BP诱导的肾和肝DNA损伤的机制。当在碳 - 2或碳 - 3处用氘取代Tris - BP时，肾DNA损伤减少；变化的幅度与Bis - BP血浆曲线下面积的减少以及每种氘代类似物的Tris - BP代谢物的肾脏共价结合的减少相关。与之形成鲜明对比的是，在碳 - 2或碳 - 3处标记有氘的Bis - BP类似物与未标记的Bis - BP相比，肾DNA损伤的严重程度并未降低。基于这些观察结果，提出了一种肝P - 450介导的Tris - BP在碳 - 2或碳 - 3处氧化生成Bis - BP的代谢方案，该过程通过两条易受初级氘动力学同位素效应影响的替代途径进行。Tris - BP代谢物Bis - BP随后代谢为导致DNA损伤并以独立于细胞色素P - 450的机制与肾脏蛋白质结合的反应性中间体。
