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.
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.
Szabó
C
C
Division of Critical Care, Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Zingarelli
B
B
O'Connor
M
M
Salzman
A L
AL
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*
D009566:Nitrates; D009243:NAD; C039642:peroxynitric acid; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases