REACTIVE OXYGEN METABOLITE-INDUCED TOXICITY TO CULTURED BOVINE ENDOTHELIAL-CELLS - STATUS OF CELLULAR IRON IN MEDIATING INJURY

We aimed to determine the status of iron in mediating oxidant-induced damage to cultured bovine aortic endothelial cells. Chromium-51-labele d cells were exposed to reaction mixtures of xanthine oxidase/hypoxant hine and glucose oxidase/glucose; these produce superoxide and hydroge n peroxide, or hydrogen peroxide, respectively. Xanthine oxidase cause d a dose dependent increase of Cr-5I release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by supero xide dismutase. Prevention of xanthine oxidase-induced damage by catal ase was blocked by an inhibitor of catalase, aminotriazole. Glucose ox idase also caused a dose-dependent increase of Cr-51 release. Glucose oxidase-induced injury, which was catalase-inhibitable, was not preven ted by extracellular superoxide dismutase. Both addition of and pretre atment with deferoxamine (a chelator of Fe3+) prevented glucose oxidas e-induced injury. The presence of phenanthroline (a chelator of divale nt Fe2+) prevented glucose oxidase-induced Cr-51 release, whereas pret reatment with the agent did not. Apotransferrin (a membrane impermeabl e iron binding protein) failed to influence damage. Neither deferoxami ne nor phenanthroline influenced cellular antioxidant defenses, or inh ibited lysis by non-oxidant toxic agents. Treatment with allopurinol a nd oxypurinol, which inhibited cellular xanthine oxidase, failed to pr event glucose oxidase injury. We conclude that (1) among the oxygen sp ecies extracellularly generated by xanthine oxidase/hypoxanthine, hydr ogen peroxide induces damage via a reaction on cellular iron; (2) defe roxamine and phenanthroline protect cells by chelating Fe3+ and Fe2+, respectively; and (3) reduction of cellular stored iron (Fe3+) to Fe2 may be a prerequisite for mediation of oxidant-induced injury, but th is occurs independently of extracellular superoxide or cellular xanthi ne oxidase-derived superoxide. (C) 1994 Wiley-Liss, Inc.*