No abstract

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.

No abstract

Interaction of heme-and non-heme iron with H202 or organic hydroperoxides is known to produce oxidative stress due to formation of oxofenyl-and oxygen-derived free radical species. Using low-temperature EPR, we demonstrated oxoferrylhemoglobin (oxoferryl-Hb) free radical species in human erythroleukemia W P . 5 cells (K562-derived etoposide-resistant subline) upon exposure to tert-butylhydroperoxide (t-BuOOH). The intensity of oxoferryl EPR signals was proportional to intracellular Hb concentrations (which were manipulated by incubating the cells with different concentrations of hemin for 24 hr), and was correlated with the cell mortality, and the amount of oxidative stress. The latter was quantitated by a novel technique based on metabolic integration of cis-parinaric acid into major classes of membrane phospholipids and their subsequent fluorescence-HPLC assay. Preincubation of the cells with an NOdonor, NOC-15, resulted in the formation of non-heme iron nitrosyl complexes as well as hexa-(R-state) and pentacoordinate (T-state) Hb-nitrosyl complexes. Nitrosylation of nonheme iron and Hb prevented t-BuOOH-induced: (i) generation of oxofenyl free radical species, (ii) oxidative stress, and (iii) cell death. Importantly, KNP.5 cells transfected with the human inducible NO synthase (iNOS) gene were also resistant to t-BuOOH-induced oxidative stress. Since NO did not protect against oxidative stress induced by a lipophillic azo-initiator of peroxyl radicals (AMVN), we conclude that protective effects of NO were due to the formation of iron-nitrosyl complexes rather than scavenging of peroxyl radicals.Pharmacology,