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
ABSTRACT The free radicals nitric oxide and superoxideanion react to form peroxynitrite (ONOO2), a highly toxicoxidant species. In vivo formation of ONOO2 has been demonstrated in shock and inflammation. Herein we provideevidence that cytotoxicity in cells exposed to ONOO2 ismediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase(PARS). Exposure to ONOO2 (100 mM to 1 mM) inhibitedmitochondrial respiration in cultured J774 macrophages andin rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1–3 h after ONOO2 exposure, andreversible, with recovery after a period of 6–24 h. Theinhibition of mitochondrial respiration was paralleled by adose-dependent increase in DNA strand breakage, reaching itsmaximum at 20–30 min after exposure to ONOO2. Weobserved a dose-dependent increase in the activity of PARS incells exposed to ONOO2. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellularrespiration in cells exposed to ONOO2. Activation of PARS byONOO2-mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by adecline of intracellular NAD1 and ATP content. 3-Aminobenzamide prevented the loss of NAD1 and ATP in cells exposedto ONOO2. In contrast, impairment of cellular respiration bythe addition of the nitric oxide donors S-nitroso-N-acetyl-DLpenicillamine or diethyltriamine nitric oxide complex, was notassociated with the development of DNA strand breaks, inconcentrations up to 1 mM, and was largely refractory toPARS inhibition. Our results suggest that DNA damage andactivation of PARS, an energy-consuming futile repair cycle,play a central role in ONOO2-mediated cellular injury.