Hydrogen peroxide mediates higher order chromatin degradation

Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H2O2), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H2O2 in respect to HOCD induction. Rat glioma C6 cells were exposed to H2O2 and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H2O2 induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H2O2 at concentrations as low as 5 muM. HOCD was mostly reversible during 4-8 h following the removal of H2O2 from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H2O2 in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H2O2. The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H2O2. Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H2O2. Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H2O2, indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H2O2. (C) 2003 Elsevier Science Ltd. All rights reserved.