Hiroi Mayo, Ogihara Tohru, Hirano Kazuya, Hasegawa Masashi, Morinobu Takao, Tamai Hiroshi, Niki Etsuo
Division of Neonatology, Department of Pediatrics, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan.
Free Radic Biol Med. 2005 Apr 15;38(8):1057-72. doi: 10.1016/j.freeradbiomed.2005.01.001.
We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.
我们之前报道过，围产期窒息婴儿脑脊液中的非蛋白结合铁（NPBI）和抗坏血酸（AA）水平显著升高。本研究表明，硫酸亚铁（FeSO4）和AA协同诱导PC12细胞凋亡，而α-生育酚和谷胱甘肽（GSH）乙酯可阻止这种凋亡。自由基损伤标志物，如邻酪氨酸、间酪氨酸和F（2α）-异前列腺素，呈逐渐上升趋势。AA和亚铁NPBI从培养基中迅速消失，但仅暴露数小时就足以触发细胞凋亡。细胞内谷胱甘肽（GSH）逐渐减少，同时谷胱甘肽二硫化物（GSSG）相应增加。GSSG、2H+/2GSH偶联的基线半电池还原电位（Ehc）为-246 mV，而-200 mV的Ehc是开启细胞凋亡的临界水平，尽管一些细胞通过细胞内GSH的短暂增加逃脱了这种命运。一旦Ehc达到约-165 mV（比基线氧化81 mV），所有细胞都失去了维持足够细胞内GSH水平的能力，随后发生凋亡。这些发现至少部分解释了铁-抗坏血酸细胞毒性的机制，即亚铁催化羟基自由基的产生并诱导脂质过氧化，之后GSH的后续消耗将Ehc提高到触发或增强凋亡级联反应的临界水平。
