Stover S K, Gushansky G A, Salmen J J, Gardiner C S
Department of Biological Sciences, University of Northern Colorado, Greeley, Colorado, 80639, USA.
Toxicol Appl Pharmacol. 2000 Oct 15;168(2):153-9. doi: 10.1006/taap.2000.9030.
The present study examined expression of gamma-glutamate-cysteine ligase (GLCL; also known as gamma-glutamylcysteine synthetase), the rate-limiting enzyme for de novo synthesis of glutathione, in the preimplantation mouse embryo. Previous studies indicated that the cleavage stage embryo is unable to synthesize glutathione de novo. It is hypothesized that GLCL mRNA and protein are not normally expressed in the cleavage stage embryo, but either glutathione depletion or oxidation may induce their expression. In untreated embryos, RT-PCR and Western blotting revealed GLCL heavy subunit (GLCL-H) mRNA and protein only at the blastocyst stage of development. Furthermore, while diethyl maleate (DEM) exposure to deplete cellular glutathione did not induce expression of GLCL-H, exposure to tertiary-butyl hydroperoxide (tBH), an oxidizing agent, resulted in significant upregulation of GLCL-H expression in two-cell embryos. Neither treatment affected expression in blastocysts. Finally, HPLC analysis confirmed that tBH-treated embryos experienced oxidative stress, as indicated by an increase in the ratio of oxidized to reduced glutathione. This oxidative stress induced de novo glutathione synthesis in the cleavage stage embryo, as demonstrated by the subsequent recovery of reduced glutathione levels following DEM-induced depletion. In the absence of tBH treatment, however, cleavage stage embryos could not recover GSH after DEM-mediated depletion. This study demonstrates that the preimplantation embryo has the capacity to upregulate glutathione synthesis in response to oxidative stress but not GSH depletion. These results suggest that, while the preimplantation embryo is well adapted to dealing with oxidative stress, it may be poorly protected from GSH-depleting toxicants.
本研究检测了谷胱甘肽从头合成的限速酶γ-谷氨酸-半胱氨酸连接酶（GLCL，也称为γ-谷氨酰半胱氨酸合成酶）在植入前小鼠胚胎中的表达。先前的研究表明，卵裂期胚胎无法从头合成谷胱甘肽。据推测，GLCL mRNA和蛋白在卵裂期胚胎中通常不表达，但谷胱甘肽耗竭或氧化可能会诱导其表达。在未处理的胚胎中，RT-PCR和蛋白质印迹仅在发育的囊胚阶段检测到GLCL重亚基（GLCL-H）mRNA和蛋白。此外，虽然暴露于马来酸二乙酯（DEM）以耗尽细胞内谷胱甘肽不会诱导GLCL-H的表达，但暴露于氧化剂叔丁基过氧化氢（tBH）会导致二细胞胚胎中GLCL-H表达显著上调。两种处理均未影响囊胚中的表达。最后，高效液相色谱分析证实，tBH处理的胚胎经历了氧化应激，这表现为氧化型谷胱甘肽与还原型谷胱甘肽的比例增加。这种氧化应激诱导了卵裂期胚胎中的谷胱甘肽从头合成，这在DEM诱导的耗竭后还原型谷胱甘肽水平随后恢复中得到了证明。然而，在没有tBH处理的情况下，卵裂期胚胎在DEM介导的耗竭后无法恢复谷胱甘肽。本研究表明，植入前胚胎有能力响应氧化应激而上调谷胱甘肽合成，但对谷胱甘肽耗竭则不然。这些结果表明，虽然植入前胚胎很好地适应了应对氧化应激，但它可能对消耗谷胱甘肽的毒物保护不足。
