title: DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.
abstract: The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.
authors.affiliation: Division of Critical Care, Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
authors.forename: C
authors.initials: C
authors.lastname: Szabó
authors.forename: B
authors.initials: B
authors.lastname: Zingarelli
authors.forename: M
authors.initials: M
authors.lastname: O'Connor
authors.forename: A L
authors.initials: AL
authors.lastname: Salzman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016923:Cell Death; D002460:Cell Line; D004249:DNA Damage; D004734:Energy Metabolism; D004789:Enzyme Activation; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology*; D009243:NAD / Q000378:metabolism; D009566:Nitrates / Q000633:toxicity*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
chemical_list: D009566:Nitrates; D009243:NAD; C039642:peroxynitric acid; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases