The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.The cellular polycationic polyamines are ubiquitous in nature and are absolutely required for eukaryotic cell growth (1, 2). However, very few specific molecular functions of polyamines have been described (3, 4). Some of the attributes ascribed to polyamines, particularly spermine, include the regulation of gene expression (5), the stabilization of chromatin (6-8), the prevention of endonuclease-mediated DNA fragmentation (9), and the inhibition of DNA damage (8, 10-13). Lovaas has recently reviewed many of these properties (14). Although protection of DNA damage by polyamines has been demonstrated, the mechanisms by which spermine functions in this protection are not known. Most of the proposals suggest that polyamine action is a result of charge neutralization and conformational changes (8,15). Because spermine is thought to be intimately associated with chromatin (7), we sought to investigate if spermine had a direct role in protecting DNA from free radical attack. Free radical and other reactive oxygen species (ROS) generation through normal cellular metabolism and by exogenous insult is a constant problem for which cells have developed multiple protective mechanisms to survive(16). Here we demonstrate that spermine, which is normally found in millimolar concentrations in the nucleus (17), can function directly as a free radical scavenger. MATERIALS AND METHODSAssays for DNA Strand Breaks. DNA strand breakage was measured by the conversion to open circular and linear forms of supercoiled ⌽X-174 RF1 double-stranded DNA (New England Biolabs). To assess DNA cleavage, 0.2 g of DNA was incubated in the presence of 30 M H 2 O 2 and 10 M CuCl 2 in PBS (pH 7.4) in a total volume of 30 l as described (18,19). Polyamines or antioxidants were coincubated as indicated. Following incubation the samples were separated by electrophoresis in a 1% agarose gel containing 40 mM Tris-acetate and 1 mM EDTA in a horizontal slab gel apparatus using Tris/acetate gel buffer. The gel was stained with ethidium bromide (2 g/ml) for 10 min, followed by destaining in water for 10 min, and was then photographed by UV translumination. The gels were photographed using an Eagle Eye digital camera (Stratagene). A single strand break in supercoiled double-stranded DNA results in the formation of open circular DNA, and double strand breaks result in the formation of linea...

␣-Thujone is the toxic agent in absinthe, a liqueur popular in the 19th and early 20th centuries that has adverse health effects. It is also the active ingredient of wormwood oil and some other herbal medicines and is reported to have antinociceptive, insecticidal, and anthelmintic activity. This study elucidates the mechanism of ␣-thujone neurotoxicity and identifies its major metabolites and their role in the poisoning process. Four observations establish that ␣-thujone is a modulator of the ␥-aminobutyric acid (GABA) type A receptor. First, the poisoning signs (and their alleviation by diazepam and phenobarbital) in mice are similar to those of the classical antagonist picrotoxinin. Second, a strain of Drosophila specifically resistant to chloride channel blockers is also tolerant to ␣-thujone. Third, ␣-thujone is a competitive inhibitor of [ 3 H]ethynylbicycloorthobenzoate binding to mouse brain membranes. Most definitively, GABA-induced peak currents in rat dorsal root ganglion neurons are suppressed by ␣-thujone with complete reversal after washout. ␣-Thujone is quickly metabolized in vitro by mouse liver microsomes with NADPH (cytochrome P450) forming 7-hydroxy-␣-thujone as the major product plus five minor ones (4-hydroxy-␣-thujone, 4-hydroxy-␤-thujone, two other hydroxythujones, and 7,8-dehydro-␣-thujone), several of which also are detected in the brain of mice treated i.p. with ␣-thujone. The major 7-hydroxy metabolite attains much higher brain levels than ␣-thujone but is less toxic to mice and Drosophila and less potent in the binding assay. The other metabolites assayed are also detoxification products. Thus, ␣-thujone in absinthe and herbal medicines is a rapid-acting and readily detoxified modulator of the GABA-gated chloride channel.

Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.rapid apoptosis ͉ caspases ͉ target identification