Iron-mediated carcinogenesis is thought to occur through the generation of oxygen radicals. Iron chelators are used in attempts to prevent the long term consequences of iron overload. In particular, 1,2-dimethyl-3- hydroxypyrid-4-one (L1), has shown promise as an effective chelator. Using an established hepatocellular model of iron overload, we studied the generation of iron-catalyzed oxidative DNA damage and the influence of iron chelators, including L1, on such damage. Iron loading of HepG2 cells was found to greatly exacerbate hydrogen peroxide-mediated DNA damage. Desferrithiocin was protective against iron/hydrogen peroxide-induced DNA damage; deferoxamine had no effect. In contrast, L1 exposure markedly potentiated hydrogen peroxide-mediated oxidative DNA damage in iron-loaded liver cells. However, when exposure to L1 was maintained during incubation with hydrogen peroxide, L1 exerted a protective effect. We interpret this as indicating that L1's potential toxicity is highly dependent on the L1:iron ratio. In vitro studies examining iron-mediated ascorbate oxidation in the presence of L1 showed that an L1:iron ratio must be at least 3 to 1 for L1 to inhibit the generation of free radicals; at lower concentrations of L1 increased oxygen radical generation occurs. In the clinical setting, such potentiation of iron- catalyzed oxidative DNA damage at low L1:iron ratios may lead to long-term toxicities that might preclude administration of L1 as an iron chelator. Whether this implication in fact extends to the in vivo situation will have to be verified in animal studies.