DNA Strand Breakage, Activation of Poly(ADP-Ribose) Synthetase, and Cellular Energy Depletion are Involved in the Cytotoxicity in Macrophages and Smooth Muscle Cells Exposed to Peroxynitrite

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 5; pp. 1753 - 1758
• Main Authors: Szabó, Csaba, Zingarelli, Basilia, O'Connor, Michael, Salzman, Andrew L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 05.03.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Adenosine Triphosphate - metabolism; Animals; Cell Death; Cell Line; Cell respiration; Cellular biology; Cultured cells; Deoxyribonucleic acid; DNA; DNA Damage; Energy Metabolism; Enzyme Activation; Enzymes; Free radicals; Hydrogen; Islet cells; Macrophages; Macrophages - physiology; Mice; Mitochondria - metabolism; Muscle, Smooth - cytology; NAD - metabolism; Nitrates - toxicity; Peroxides; Poly(ADP-ribose) Polymerases - metabolism; Respiration; Superoxides; Toxicity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.5.1753

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 μ M 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.