ACTIVE OXYGEN-MEDIATED CHROMOSOMAL 1-2 MBP GIANT DNA FRAGMENTATION INTO INTERNUCLEOSOMAL DNA FRAGMENTATION IN APOPTOSIS OF GLIOMA-CELLS INDUCED BY GLUTAMATE

C6 glioma cells treated with 10 mM glutamate reduced intracellular GSH to one-seventh of the initial level, and induced cytolysis accompanie d by apoptosis. The treated cells produced extracellular H2O2. The cyt olysis of the C6 cells induced by glutamate was prevented by antioxida nts such as N-acetylcysteine (NAC), ascorbic acid (ASC), catalase, and NaN3, iron chelators such as deferoxamine and 1,10-phenanthroline, an d oxygen radical scavengers such as 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-phenyl-tert-butyl nitrone (PEN). The effect of these antioxidants, iron chelators, and oxygen radical scavengers on the cyt olysis of C6 cells was dependent on the dose and the intracellular GSH level. Furthermore, 1-2 Mbp chromosomal DNA (giant DNA) fragments wer e observed during cytolysis. The giant DNA fragments were further clea ved into smaller DNA fragments of 200-800 kbp, and then to fragments o f less than 300 kbp in size including chromosomal ladder DNA fragments . Such serial chromosomal DNA degradations induced by glutamate were a lso inhibited by addition of these antioxidants, iron chelators, and o xygen radical scavengers, These findings suggest that glutamate induce s GSH depletion, and consequently, apoptosis through endogenously prod uced active oxygen species in C6 glioma cells and that the apoptosis i s accompanied by 1-2 Mbp giant DNA fragmentation prior to the internuc leosomal DNA fragmentation. (C) 1998 Elsevier Science Inc.