To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D, Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 ,uM H202 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H202. H202 at 40 MuM or higher inhibited DNA synthesis, and induced SSB within less than 30 s.These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.

H202, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis oftarget cells. This may be an important aspect of inflammatory iD'jury of tissues. Cell lysis in two target cells, the murine macrophagelike tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADPribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADPribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H202 prevented the sequence of events that eventually led to cell lysis-i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADPribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks.Oxidants induce cell injury and lysis in various target cells both in vivo and in vitro (1)(2)(3)(4) ], a nuclear enzyme using NAD as its substrate and forming nicotinamide and poly(ADP-ribose) polymers, which are ester-bound to various nuclear proteins. Poly-(ADP-ribose) polymerase activation has been associated with conditions that cause DNA damage (9-12) and results in depletion of NAD in irradiated cells (9) and cells exposed to alkylating agents (10, 11). Poly(ADP-ribose) polymerase can be inhibited by 3-aminobenzamide (which is considered to be specific), nicotinamide, theophylline, and high doses of thymidine (13).The effect of inhibition of poly(ADP-ribose) polymerase on H202-induced cellular injury is not known. Since this enzyme appears to exert a central function in the biochemical chain of events following exposure of cells to the oxidant H202, it is important to know whether its action is essential to the development of injury to the target cell. Therefore, NAD, ATP, cellular Ca2+, actin polymerization, and cell lysis as parameters of oxidant-induced cell injury were determined in the presence or absence of inhibitors of poly(ADP-ribose) polymerase. MATERIALS AND METHODSCell Culture. P388D1 cells were cultured in RPMI 1640 medium (Irvine Scientific) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 ,ug of gentamycin sulfate (M. A. Bioproducts, Walkersville, MD) per ml.

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Little is known about the biochemical "machinery" responsible for the morphological features of apoptosis, although the cytoskeleton is presumed to be involved. Using flow cytometry, polyacrylamide gel electrophoresis, and fluorescence microscopy, we show that apoptosis induced by ultraviolet (UV) irradiation or 80 micrograms/ml etoposide correlates with early transient polymerization and later depolymerization of filamentous (F)-actin and dramatic changes in visible microfilament organization. Depolymerization of F-actin began before the formation of apoptotic bodies and was ultimately composed of decreases in both the detergent-insoluble (40%) and detergent-soluble (50%) pools of F-actin. Dihydrocytochalasin B (H2CB), which blocked apoptotic body formation, depolymerized F-actin in the detergent-insoluble pool only. Visually, H2CB treatment disrupted microfilament organization, resulting in short, brightly stained microfilaments dispersed throughout the cytoplasm. In contrast, apoptotic cells contained a network of fine microfilaments with bright staining concentrated at the site of apoptotic body formation. Together, these results suggest that reorganization of the microfilament network is necessary for the formation of apoptotic bodies and that depolymerization of F-actin may also be a necessary component of the process of apoptosis.