Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid.
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.
Hiroi
Mayo
M
Division of Neonatology, Department of Pediatrics, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan. ped025@poh.osaka-med.ac.jp
Ogihara
Tohru
T
Hirano
Kazuya
K
Hasegawa
Masashi
M
Morinobu
Takao
T
Tamai
Hiroshi
H
Niki
Etsuo
E
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism*; D007501:Iron / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats
D007501:Iron; D005978:Glutathione; D001205:Ascorbic Acid