Rundgren M, Porubek D J, Harvison P J, Cotgreave I A, Moldéus P, Nelson S D
Department of Toxicology, Karolinska Institute, Stockholm, Sweden.
Mol Pharmacol. 1988 Oct;34(4):566-72.
N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, has previously been shown to be toxic to hepatocytes freshly isolated from rat liver [Mol. Pharmacol. 28:306-311 (1985)] NAPQI arylates and oxidizes cellular thiols, and either one or both reactions may be important in the pathogenesis of cytotoxicity. Two dimethylated analogues of NAPQI, N-acetyl-3,5-dimethyl-p-benzoquinone imine (3,5-diMeNAPQI) and N-acetyl-2,6-dimethyl-p-benzoquinone imine (2,6-diMeNAPQI), were prepared to determine whether one reaction might be more damaging to cells than the other. Of the three quinone imines, the least potent cytotoxin to rat hepatocytes was 3,5-diMeNAPQI. However, the cytotoxicity of 3,5-diMeNAPQI was markedly enhanced by pretreatment of cells with 1,3-bis-(2-chloroethyl)-N-nitrosourea, which inhibits glutathione reductase. Reactions of 3,5-diMeNAPQI with GSH, both chemically and in hepatocytes, indicated that this quinone imine primarily oxidized thiols. These findings were corroborated by results of covalent binding experiments, which showed that radiolabeled 3,5-diMeNAPQI bound only to a small extent to hepatocyte proteins. On the other hand, 2,6-diMeNAPQI, the most potent cytotoxin of the three quinone imines that was investigated bound extensively to hepatocyte proteins. In addition, 2,6-diMeNAPQI reacted with GSH, both chemically and in hepatocytes, to form significant amounts of GSSG. Reduction products of NAPQI and its dimethylated analogues were not important contributors to cytotoxicity or GSSG formation based on the following results: 1) the quinone imines did not increase oxygen consumption by hepatocytes nor did they lead to oxygen uptake in solution; 2) dicoumarol, an inhibitor of the reductase, DT-diaphorase, had no effect on cytotoxicity caused by the quinone imines. Evidence for the involvement of ipso-adducts of the quinone imines in their reactions with cellular thiols is provided by results of investigations on the effects of DTT on the metabolism, covalent protein binding, and cytotoxic effects of the quinone imines.
对乙酰氨基酚的活性代谢产物N-乙酰对苯醌亚胺（NAPQI），先前已被证明对从大鼠肝脏中新鲜分离的肝细胞有毒性[《分子药理学》28:306 - 311（1985）]。NAPQI使细胞硫醇芳基化并氧化，这两种反应中的一种或两种在细胞毒性的发病机制中可能都很重要。制备了NAPQI的两种二甲基化类似物，N-乙酰-3,5-二甲基对苯醌亚胺（3,5-二甲基NAPQI）和N-乙酰-2,6-二甲基对苯醌亚胺（2,6-二甲基NAPQI），以确定一种反应是否可能比另一种反应对细胞的损害更大。在这三种醌亚胺中，对大鼠肝细胞毒性最小的是3,5-二甲基NAPQI。然而，用抑制谷胱甘肽还原酶的1,3-双（2-氯乙基）-N-亚硝基脲预处理细胞后，3,5-二甲基NAPQI的细胞毒性显著增强。3,5-二甲基NAPQI与谷胱甘肽（GSH）在化学和肝细胞中的反应表明，这种醌亚胺主要氧化硫醇。共价结合实验结果证实了这些发现，该实验表明放射性标记的3,5-二甲基NAPQI仅在很小程度上与肝细胞蛋白结合。另一方面，2,6-二甲基NAPQI是所研究的三种醌亚胺中毒性最强的，它与肝细胞蛋白广泛结合。此外，2,6-二甲基NAPQI在化学和肝细胞中与GSH反应，形成大量的氧化型谷胱甘肽（GSSG）。基于以下结果，NAPQI及其二甲基化类似物的还原产物对细胞毒性或GSSG形成的贡献不大：1）醌亚胺既不增加肝细胞的耗氧量，也不导致溶液中的氧摄取；2）还原酶DT-黄递酶的抑制剂双香豆素对醌亚胺引起的细胞毒性没有影响。对二硫苏糖醇（DTT）对醌亚胺的代谢、共价蛋白结合和细胞毒性作用的影响的研究结果，为醌亚胺的本位加合物参与其与细胞硫醇的反应提供了证据。
