Kassahun K, Jochheim C M, Baillie T A
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle 98195.
Biochem Pharmacol. 1994 Aug 3;48(3):587-94. doi: 10.1016/0006-2952(94)90290-9.
The present study examined the effects of S-(N-methylcarbamoyl)glutathione (SMG), S-(N-methylcarbamoyl)-L-cysteine (L-SMC) and some analogs of these S-linked conjugates of methyl isocyanate (MIC) on the activity of glutathione reductase (GR) in freshly isolated rat hepatocytes and on the levels of reduced and oxidized glutathione (GSH and GSSG) in exposed cells. Both SMG and its monoethyl ester (0.5 mM) were found to inhibit GR weakly, although L-SMC proved to be an effective inhibitor of the enzyme (60 +/- 4% activity remaining after a 4-hr incubation at 0.5 mM). The cysteine adduct (SCC) of 2-chloroethyl isocyanate (CEIC) was a strong inhibitor of GR (27 +/- 1% activity remaining after a 1-hr incubation at 0.1 mM) and was essentially equipotent with the antitumor agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). L-SMC depleted intracellular GSH in a time- and concentration-dependent manner up to 2 hr of incubation, beyond which time GSH levels began to recover. Exposure of cells to the enantiomeric conjugate, D-SMC, led to a similar concentration- and time-dependent inhibition of GR and fall in intracellular GSH, but in this case the depletion of GSH was extensive and was sustained throughout the 5-hr incubation period. Only a small amount (less than 10%) of the GSH that was lost from cells exposed to SMC was recovered in the medium, indicating that SMC did not cause efflux of GSH (most of the free cysteine released during breakdown of SMC was recovered in the medium). Experiments with hepatocytes exposed for 5 hr to SCC (0.1 mM) demonstrated that GSSG levels were elevated by 32 +/- 5% relative to controls. Collectively, these results indicate that carbamate thioester conjugates of MIC and CEIC inhibit GR, probably via release of the free isocyanate at the cell surface, which then penetrates the hepatocyte. The inhibitory effects of the isocyanates on GR, coupled with their propensity to react spontaneously with GSH, combine to deplete significantly intracellular stores of GSH.
本研究考察了S-(N-甲基氨基甲酰基)谷胱甘肽(SMG)、S-(N-甲基氨基甲酰基)-L-半胱氨酸(L-SMC)以及这些异氰酸甲酯(MIC)的S-连接共轭物的一些类似物对新鲜分离的大鼠肝细胞中谷胱甘肽还原酶(GR)活性以及对暴露细胞中还原型和氧化型谷胱甘肽(GSH和GSSG)水平的影响。发现SMG及其单乙酯(0.5 mM)对GR有微弱抑制作用，尽管L-SMC被证明是该酶的有效抑制剂(在0.5 mM下孵育4小时后，活性保留60±4%)。2-氯乙基异氰酸酯(CEIC)的半胱氨酸加合物(SCC)是GR的强抑制剂(在0.1 mM下孵育1小时后，活性保留27±1%)，且基本上与抗肿瘤药物N,N'-双(2-氯乙基)-N-亚硝基脲(BCNU)等效。L-SMC在长达2小时的孵育时间内以时间和浓度依赖的方式消耗细胞内的GSH，超过该时间后GSH水平开始恢复。将细胞暴露于对映体共轭物D-SMC会导致类似的浓度和时间依赖性的GR抑制以及细胞内GSH下降，但在这种情况下，GSH的消耗广泛且在整个5小时的孵育期内持续存在。暴露于SMC的细胞中损失的GSH只有少量(不到10%)在培养基中回收，这表明SMC不会导致GSH外流(SMC分解过程中释放的大部分游离半胱氨酸在培养基中回收)。用肝细胞在0.1 mM SCC下暴露5小时的实验表明，相对于对照，GSSG水平升高了32±5%。总体而言，这些结果表明，MIC和CEIC的氨基甲酸酯硫酯共轭物可能通过在细胞表面释放游离异氰酸酯来抑制GR，然后游离异氰酸酯穿透肝细胞。异氰酸酯对GR的抑制作用，再加上它们与GSH自发反应的倾向，共同导致细胞内GSH储备显著耗尽。
