Fariss M W, Bryson K F, Tirmenstein M A
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman 99164-6510, USA.
Biochem Pharmacol. 1997 Mar 7;53(5):651-61. doi: 10.1016/s0006-2952(96)00874-x.
Suspensions of rat hepatocytes treated with the alkylating agent ethyl methanesulfonate (EMS) exhibited extensive lipid peroxidation as well as rapid and near complete depletion of cellular reduced glutathione (GSH) levels prior to cell death. Pretreatment of hepatocytes with medium deficient in sulfur amino acids accelerated cell death induced by EMS, confirming the previously reported cytoprotective role for GSH in this toxic event. Nearly all of the cellular GSH lost following 50 mM EMS treatment was accounted for as S-ethyl glutathione (GS-Et). No significant formation of glutathione disulfide was observed. The GS-Et formed was not exported from the cell but remained at high intracellular concentrations throughout the course of the experiment. In addition, EMS treatment inhibited the efflux of intracellular GSH and inhibited the cellular accumulation of glutamate (Glu). Supplementation of hepatocytes with 25 microM d-alpha-tocopheryl hemisuccinate (TS) protected these cells against EMS-induced lipid peroxidation and cell death. Cytoprotection with TS had no effect on EMS-induced depletion of intracellular GSH or intracellular levels of GS-Et or Glu. However, TS supplementation did prevent EMS-induced depletion of cellular protein thiols. Interestingly, the pretreatment of hepatocytes with 1 mM dithiothreitol promoted EMS toxicity. The results of this study suggest that the cytoprotective abilities of TS are related to the prevention of both EMS-induced lipid peroxidation and protein thiol depletion. Thus, the onset of lipid peroxidation and the loss of protein thiols in hepatocytes appear to be critical cellular events leading to EMS-induced cell death.
用烷化剂甲磺酸乙酯（EMS）处理的大鼠肝细胞悬液在细胞死亡前表现出广泛的脂质过氧化以及细胞内还原型谷胱甘肽（GSH）水平迅速且近乎完全耗尽。用缺乏含硫氨基酸的培养基预处理肝细胞会加速EMS诱导的细胞死亡，证实了先前报道的GSH在这一毒性事件中的细胞保护作用。50 mM EMS处理后几乎所有细胞内损失的GSH都以S - 乙基谷胱甘肽（GS - Et）的形式存在。未观察到明显的谷胱甘肽二硫化物形成。形成的GS - Et并未从细胞中排出，而是在整个实验过程中保持高细胞内浓度。此外，EMS处理抑制了细胞内GSH的外流，并抑制了谷氨酸盐（Glu）的细胞内积累。用25 microM d-α-生育酚半琥珀酸酯（TS）补充肝细胞可保护这些细胞免受EMS诱导的脂质过氧化和细胞死亡。TS的细胞保护作用对EMS诱导的细胞内GSH耗尽或GS - Et或Glu的细胞内水平没有影响。然而，补充TS确实能防止EMS诱导的细胞蛋白巯基的耗尽。有趣的是，用1 mM二硫苏糖醇预处理肝细胞会促进EMS毒性。本研究结果表明，TS的细胞保护能力与预防EMS诱导的脂质过氧化和蛋白巯基耗尽有关。因此，肝细胞中脂质过氧化的发生和蛋白巯基的丧失似乎是导致EMS诱导细胞死亡的关键细胞事件。
