Liu Y Y, Lu A Y, Stearns R A, Chiu S H
Department of Animal and Exploratory Drug Metabolism, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Chem Biol Interact. 1992 Mar;82(1):1-19. doi: 10.1016/0009-2797(92)90010-i.
When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)
当以1、10或50毫克/千克体重的剂量给大鼠腹腔注射单剂量的[14C]三硝基甲苯时，在珠蛋白、血浆蛋白以及肝脏和肾脏中的蛋白质中检测到了共价结合的放射性。共价结合的程度呈剂量依赖性，在给药后4小时内，血浆和肾脏蛋白中的共价结合程度始终最高。然而，珠蛋白中共价加合物水平的下降比其他蛋白慢。在50毫克/千克体重的剂量下，珠蛋白共价加合物水平在给药后1小时达到峰值，为182皮摩尔/毫克蛋白，随后在第1天至第8天之间降至约50皮摩尔/毫克蛋白。在肝脏和肾脏的共价加合物水平中，发现10000×g和微粒体部分中的加合物水平高于胞质部分。与珠蛋白和肝脏蛋白共价结合的放射性易受稀酸水解的影响，通过溶剂萃取回收的主要产物是2-氨基-4,6-二硝基甲苯（2A）和4-氨基-2,6-二硝基甲苯（4A）。乙酰化后，水解产物产生的衍生物经质谱分析和与标准样品的高效液相色谱共色谱分析鉴定为2A和4A的乙酸酯。在以50毫克/千克体重腹腔注射[14C]TNT 4小时后，发现约0.4%的剂量以与血红蛋白的结合加合物形式存在，其中约48%在酸水解后作为可溶剂萃取的放射性物质回收。约2%的放射性剂量存在于肝脏中，其中约30%与肝脏蛋白共价结合，其中约49%在酸水解后可转化为可溶剂萃取的放射性物质。[14C]TNT与血液的体外孵育表明，孵育的前2小时内珠蛋白中共价加合物呈线性增加；共价加合物的浓度略高于血浆蛋白中的浓度。从珠蛋白加合物水解产物中回收的主要化合物也是体内研究中从珠蛋白中获得的2A和4A。基于体外和体内研究结果，我们证实了以1、10或50毫克/千克体重给大鼠单次腹腔注射[14C]TNT或通过与血液进行体外孵育后会形成蛋白质加合物。（摘要截于400字）
