IDENTIFICATION OF A MECHANISM OF IRON UPTAKE BY CELLS WHICH IS STIMULATED BY HYDROXYL RADICALS GENERATED VIA THE IRON-CATALYZED HABER-WEISSREACTION

Recent studies have demonstrated that preincubation of SK-Mel-28 melan oma cells with ferric ammonium citrate (FAC) resulted in marked stimul ation of Fe-59 uptake from Fe-59-I-125-transferrin (Tf), but only at T f concentrations above that required for saturation of the Tf receptor (Richardson and Baker (1992) J. Biol. Chem. 267, 13972-13979). The me chanism responsible for this stimulation was unknown and is the subjec t of the present report. Preincubation of cells with FAC (25 mu g/ml), followed by a 2 h incubation with Fe-59-I-125-Tf(0.1 mg/ml; 1.25. mu M), resulted in temperature-dependent Fe-59 uptake to approx, 200% of the control value. Furthermore, the effect was not specific for melano ma cells and was also observed in other normal and neoplastic cells. P reincubation of melanoma cells with FAC also stimulated Fe-59 uptake f rom Fe-59-citrate, but to a far greater extent than that observed with Fe-59-I-125-Tf(viz., > 20-fold that seen for the control). Interestin gly, neither receptor-mediated endocytosis nor the postulated diferric Tf reductase were involved in the FAC-activated Fe uptake process fro m Tf. However, the addition of free radical scavengers to FAC such as catalase, superoxide dismutase, ceruloplasmin, Hepes, mannitol and hig h concentrations of BSA or ascorbate, markedly depressed FAC-activated Fe-59 uptake from Fe-59-I-125-Tf and Fe-59-citrate. These agents when added to control cells had no effect on Fe-59 uptake. The addition of superoxide generating agents and hydrogen peroxide to minimum essenti al medium (MEM) containing FAC but not to MEM alone, also stimulated F e uptake. These data suggest that the initial activation of the FAC-st imulated Fe uptake system was caused by the production of hydroxyl rad icals via the Fe-catalysed Haber-Weiss reaction. We propose that this Fe uptake process represents an important cellular defense mechanism a gainst oxidant stress generated in the presence of low-molecular-weigh t Fe complexes.