Leung K H, Yao M, Stearns R, Chiu S H
Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065, USA.
Chem Biol Interact. 1995 Jun 30;97(1):37-51. doi: 10.1016/0009-2797(94)03606-9.
Studies were undertaken to investigate the mechanism of bioactivation and covalent binding of TNT. Incubation of [14C]TNT with rat liver microsomes in the presence of an NADPH generating system resulted in metabolism and covalent binding to microsomal proteins. Time-dependence studies showed that TNT was rapidly reduced to yield 4-hydroxylamino-2,6-dinitrotoluene (4HA), 4-amino-2,6-dinitrotoluene (4A) and 2-amino-4,6-dinitrotoluene (2A) as intermediates which were further metabolized to form 2,4-diamino-6-nitrotoluene (2,4DA) and 2,6-diamino-4-nitrotoluene (2,6DA). In contrast to the rapid disappearance of TNT, formation of covalent protein adducts increased with time, suggesting that the reactive intermediate was likely to be formed not directly from TNT but from proximal intermediates such as 4HA. The hypothesis that 4HA was more readily converted to the reactive intermediate than TNT was further supported by the increased levels of covalent adduct formation when [14C]4HA was incubated directly with liver microsomes. Covalent binding of TNT and 4HA was dependent on oxygen concentration. Higher levels of covalent adducts were formed when TNT was incubated aerobically (up to 50% oxygen concentration) than under anaerobic conditions. Covalent binding of [14C]4HA also increased with increasing oxygen concentrations. These results suggest that the reactive intermediate is likely to be an oxidized metabolite of 4HA, e.g. 4-nitroso-2,6-dinitrotoluene. Compounds containing a free sulfhydryl group (cysteine, N-acetylcysteine, GSH or 3,4-dichlorobenzenethiol) decreased the amount of covalent binding to various degrees, suggesting the involvement of the sulfhydryl group in adduct formation with TNT following bioactivation. Metabolic activation of TNT by liver microsomes required NADPH but not NADH as the cofactor. Incubation of [14C]TNT with purified rat liver NADPH cytochrome P450 reductase under either aerobic or anaerobic conditions yielded exclusively 4HA. In contrast, 2A and 4A were formed following incubation of TNT with the reconstituted system containing cytochrome P450, NADPH cytochrome P450, reductase and dilauroyl phosphatidylcholine. These observations suggest that the initial reduction of the nitro group can be catalyzed by NADPH cytochrome P450 reductase alone but cytochrome P450 is needed in the reduction of the hydroxylamine to the amine.
开展了多项研究以探究三硝基甲苯（TNT）的生物活化及共价结合机制。在存在NADPH生成系统的情况下，将[¹⁴C]TNT与大鼠肝脏微粒体一起孵育，会导致其代谢并与微粒体蛋白发生共价结合。时间依赖性研究表明，TNT迅速被还原，生成4-羟基氨基-2,6-二硝基甲苯（4HA）、4-氨基-2,6-二硝基甲苯（4A）和2-氨基-4,6-二硝基甲苯（2A）作为中间体，这些中间体进一步代谢形成2,4-二氨基-6-硝基甲苯（2,4DA）和2,6-二氨基-4-硝基甲苯（2,6DA）。与TNT的快速消失相反，共价蛋白加合物的形成随时间增加，这表明反应性中间体可能不是直接由TNT形成，而是由诸如4HA等近端中间体形成。当[¹⁴C]4HA直接与肝脏微粒体孵育时，共价加合物形成水平增加，进一步支持了4HA比TNT更容易转化为反应性中间体的假设。TNT和4HA的共价结合取决于氧气浓度。当TNT在需氧条件下（氧气浓度高达50%）孵育时，形成的共价加合物水平高于厌氧条件下。[¹⁴C]4HA的共价结合也随氧气浓度增加而增加。这些结果表明，反应性中间体可能是4HA的氧化代谢物，例如4-亚硝基-2,6-二硝基甲苯。含有游离巯基的化合物（半胱氨酸、N-乙酰半胱氨酸、谷胱甘肽或3,4-二氯苯硫酚）在不同程度上减少了共价结合量，这表明巯基参与了生物活化后与TNT形成加合物的过程。肝脏微粒体对TNT的代谢活化需要NADPH作为辅因子，而不需要NADH。在需氧或厌氧条件下，将[¹⁴C]TNT与纯化的大鼠肝脏NADPH细胞色素P450还原酶一起孵育，仅产生4HA。相反，将TNT与含有细胞色素P450、NADPH细胞色素P450还原酶和二月桂酰磷脂酰胆碱的重组系统一起孵育后，会形成2A和4A。这些观察结果表明，硝基的初始还原可单独由NADPH细胞色素P450还原酶催化，但将羟胺还原为胺需要细胞色素P450。
