Reactive oxygen species participate in peroxynitrite-induced apoptosis in HL-60 cells

Abstract

Peroxynitrite (ONOO-) is a physiological product generated by the interaction of superoxide (O2.-) and nitric oxide (.NO). We have previously shown that peroxynitrite induces apoptosis in HL-60 cells. In the present study, we demonstrated that peroxynitrite generates reactive oxygen species (ROS) in HL-60 cells. Brief exposure of HL-60 cells to ONOO- induced elevation of lucigenin chemiluminescence, indicating generation of superoxide anion. Exogenous superoxide dismutase (SOD), a scavenger of O2.-, fully abolished the chemiluminescence response, further supporting this notion. Following O2.- generation, the accumulation of hydrogen peroxide (H2O2) was observed. The addition of SOD exacerbated but that of catalase attenuated peroxynitrite-induced DNA fragmentation, suggesting that this H2O2 production contributes to the apoptotic process. In addition, pre-treatment of HL-60 cells with N-acetyl-L-cysteine (15 mM), a ROS scavenger, fully scavenged peroxynitrite-elicited ROS generation and effectively inhibited (ONOO-)-induced apoptosis, further enforcing this hypothesis. In summary, our results suggest that (ONOO-)-stimulated ROS formation may serve as a mechanism for the propagation of peroxynitrite-mediated apoptotic cell death in an intact cell system.