These results are consistent with previous studies demonstrating that pharmacologic concentrations of AA exert effects through the production of ROS and H2O2 and the corresponding cellular toxicity imposed by this oxidative stress. The production of H2O2 initiated by 2-Pyridylethylamine dihydrochloride treatment with AA has been proposed to occur via an extracellular mechanism both in vitro and in vivo. In combination-treated H1299 cells, ROS production was rapidly induced with significantly higher levels than control or the single treatments observed as early as 15 min after treatment. Both AA and 3-PO individually induced some ROS production; however, the maximum levels observed were 4- to 7-fold less than that observed in the combination treatment. ROS levels in lung epithelial cells were not increased by treatment with either AA or 3-PO. The combination treatment did induce a modest increase in ROS levels in lung epithelial cells after 4 h; however, this ROS induction was 7-fold less than that induced in H1299 cells. Based on previously published studies, it is likely that the induction of ROS in combination-treated cells resulted in the significant H2O2 accumulation. Our observations that addition of the impermeant H2O2 scavenger catalase to the medium reduced combinationinduced cell death in NSCLC cells by as much as 70% and that combination-induced cell death was enhanced by inhibition of endogenous catalase by aminotriazole are consistent with this hypothesis. The inability to completely prevent combinationinduced cell death by catalase is likely due to the fact that H2O2 easily crosses the plasma membrane and a portion of the H2O2 escapes degradation. It is also possible that other cytotoxic mechanisms unrelated to H2O2 are also induced by the combination treatment. Further studies of the mechanism of cell death induced by the combination of AA and 3-PO clearly demonstrated that the induction of apoptosis was a key component of this response. Significantly higher levels of DNA fragmentation were observed in combination-treated cells compared to control cells and cells treated with either AA or 3-PO individually. Significant DNA fragmentation was observed in combination-treated cells as early as 2 h after treatment and increased rapidly until 12 h, where upon DNA fragmentation began to plateau. The level of DNA fragmentation observed in the combination-treated cells was significantly higher than the predicted sum effects of the individual treatments and paralleled the synergistic induction of cell death. PARP cleavage, 22-Oxacalcitriol another marker for apoptosis indicative of caspase activation, was also significantly higher in combination-treated cells compared to control cells and cells treated with either AA or 3-PO individually.