Nitric oxide-releasing acetylsalicylic acid (NO-ASA; NO-aspirin) developed as an anti-inflammatory agent that was expected to avoid some of the adverse effects of aspirin (ASA), was recently shown to be cytotoxic to cells of different tumor lines. The cytotoxic properties and potency of NO-ASA are different than those of ASA which implies that the intracellular targets for NO-ASA and ASA, and their mechanism of action, are different. The aim of the present study was to reveal whether the cytotoxicity induced by NO-ASA is mediated by damage to DNA. We observed that even brief (1 h) treatment of human B-lymphoblastoid TK6 cells with >or=5 microM NO-ASA led to DNA damage revealed by the alkaline and neutral comet assays, histone H2AX phosphorylation on Ser 139, and ATM phosphorylation on Ser 1981, a marker of activation of this kinase. The induction of H2AX phosphorylation was preferential to S-phase cells. Exposure to >or=5 microM NO-ASA for over 3 h led to apoptosis, also preferentially of S-phase cells. Apoptosis was atypical; while chromatin was highly condensed there was no evidence of nuclear fragmentation nor were the cells positive in the TUNEL assay though they did express activated caspase-3. The induction of phosphorylation of H2AX on Ser 139 by NO-ASA was markedly attenuated in the presence of N-acetyl-L-cysteine, a scavenger of reactive oxygen species (ROS). The data imply that the NO-ASA induces DNA damage through oxidative stress; the oxidation-generated lesions provide a signal for induction of H2AX phosphorylation during DNA replication, perhaps when the progressing replication forks collide with the primary lesions converting them to DNA double-strand breaks. Because neither induction of H2AX phosphorylation nor apoptosis were observed at equimolar concentrations of ASA, the NO moiety attached to ASA appeared to mediate these effects.