It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kB activation. This hypothesis is mainly based on the ®ndings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the af®nity of receptor to TNF. PDTC inhibits the IkB± 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-kB signaling, but instead lower the magnitude of its activation.

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When supercoiled plasmid DNA was incubated with 2,2'-azobis (2-amidinopropane)hydrochloride (AAPH) at pH 7.4 in the presence and absence of oxygen, the DNA single strands were effectively cleaved. The breaking in the presence of oxygen was not inhibited by superoxide dismutase and catalase, but inhibited by mannitol, ethanol, butyl hydroxyanisole, thiol compounds, tertiary amines and spin trapping agents N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The breaking in the absence of oxygen was inhibited by ethanol, a tertiary amine and PBN. By electron spin resonance spin-trapping with PBN, the carbon-centered radical was detected both in the presence and the absence of oxygen. Hydroxyl radical was detected by use of DMPO only in the presence of oxygen. The DNA breaking activity of AAPH was found to be due primarily to the aliphatic carbon-centered radical. While the reactivity of carbon-centered radicals have received little attention, the aliphatic carbon-centered radical generated from AAPH was found to be highly reactive to break the DNA strands.