Hydrogen peroxide insult in cultured mammalian cells: relationships between DNA single-strand breakage, poly(ADP-ribose) metabolism and cell killing

Abstract

We examined the effect of exposure to H2O2 at 37 degrees C on Chinese hamster ovary cell survival, DNA single-strand break (SSB) induction and rejoining, and activation of poly(ADP-ribose) (ADPR) polymerase. The effect of the ADPR polymerase inhibitor 3-aminobenzamide on each of these processes was also determined. SSB induction increased progressively with increasing H2O2 concentration. SSB levels were maximal after approx. 5 min of exposure to H2O2 (100 microM) and then decreased at longer times. This decrease, which paralleled the time-dependent depletion of H2O2, was due to the rejoining of SSBs. 3-Aminobenzamide enhanced the level of SSBs at each time point. H2O2 increased the level of both ADPR synthesis and NAD+ depletion (both measures of ADPR polymerase activity) in a concentration-dependent fashion, with the maximum effect being reached after approx. 20 min. After 100 microM H2O2, the effects on both ADPR and NAD+ were reversible. 3-Aminobenzamide completely blocked the effects of the oxidant on both NAD+ and ADPR levels. Thus, SSB induction by H2O2 at 37 degrees C was accompanied by a marked but reversible stimulation of ADPR polymerase. However, cell killing by H2O2 was only slightly enhanced in the presence of 3-aminobenzamide (5 mM), so the above-mentioned effects do not appear to be relevant to the cytotoxic effect of H2O2 under these conditions. Comparing these results with data obtained previously for cells treated with H2O2 at 4 degrees C suggests that the mechanisms of DNA strand breakage and cell killing may be quite different at the two temperatures, and that DNA damage at 37 degrees C may be indirectly mediated by temperature-dependent metabolic events.