In view of the current speculation regarding the possible role of
reactive oxygen species (ROS) in apoptosis, both under physiological conditions and in response to chemicals that promote their intracellular formation, the present investigation was undertaken to examine whether DNA fragmentation during oxidative stress results from
endonuclease activity (apoptosis) or from direct attack by ROS. We report that the incubation of HepG2 cells (a human-derived
hepatoma cell line) with the
copper(II) complex of
1,10-phenanthroline, CuII(OP)2, results in internucleosomal DNA fragmentation, which is widely recognized as being a hallmark of apoptosis. DNA fragmentation did not occur at low temperature, but activity was restored by the addition of
ascorbic acid. It is proposed that DNA fragmentation results from the direct attack of
hydroxyl radicals upon
DNA.
Hydroxyl radicals are produced from
oxygen by the redox-cycling of CuII(OP)2, which is supported by metabolic processes at normal temperature. At low temperature
ascorbic acid provides an artificial cellular reducing environment, thereby restoring
hydroxyl radical formation. These findings were confirmed by the detection of internucleosomal DNA fragmentation following the exposure of isolated
chromatin to a biomimetic CuII(OP)2 redox-cycling system. We conclude that
DNA laddering, the widely employed hallmark of apoptosis, is not unique to
endonuclease activity and may also result from direct attack upon
DNA by the
hydroxyl radical.