Evidence that reactive oxygen species do not mediate NF-κB activation

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)‐κB activation. This hypothesis is mainly based on the findings that N‐acetyl‐L‐cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF‐κB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF‐κB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)‐induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IκB–ubiquitin ligase activity in the cell‐free system where extracellular stimuli‐regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF‐κB signaling, but instead lower the magnitude of its activation.