Iron loading of endothelial cells augments oxidant damage

Transition metals, particularly iron, will potentiate oxidant damage to isolated cell organelles, plasma membranes, and DNA when added to in vitro incubation systems. However, similar studies of intact cells have been hampered by the relative impermeability of whole cells to iron. We have iron loaded cultured endothelial cells by using the iron-chelating fungistat 8-hydroxyquinoline (8HQ). 8HQ forms lipophilic chelates with iron and rapidly transfers the metal across the intact plasma membrane of endothelial cells. After brief exposure to 8HQ and subsequent thorough washing of endothelial cells, the cell-associated iron cannot be removed by the powerful chelator deferoxamine, clearly indicating the intracellular location of 8HQ-transported iron. Iron-loaded cells (but not cells exposed to high concentrations of 8HQ or iron separately) are extremely sensitive to oxidants (1) produced externally by phorbol-stimulated granulocytes, (2) generated intracellularly by menadione, or (3) added as H₂O₂. In the latter instance, as little as 7 μmol/L H₂O₂ provokes destruction of ~50% of iron-loaded endothelial cells, whereas untreated endothelium readily survives exposure to H₂O₂ concentrations as high as 2 mmol/L. Cytotoxicity is accompanied by membrane lipid peroxidation (formation of thiobarbituric acid-reactive substances). Both cytotoxicity and lipid peroxidation are inhibited by the lipophilic 21-aminosteroid U74500A ("lazaroid") (50% inhibitory concentration = approximately 0.5 μmol/L), whereas deferoxamine (250 μmol/L) is ineffective (suggesting iron intercalation into hydrophobic domains of the cell). We conclude that this pharmacologic model for iron loading of intact cells may yield valuable insights into the pathogenic importance of intracellular iron in iron overload states, inflammation, and cellular injury.