Elevated Ferritin Production, Iron Containment, and Oxidant Resistance in Hemin-Treated Leukemia Cells

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemoglobin, is a source of potentially cytotoxic iron, but in chronic low doses can induce cytoprotection against iron-stimulated oxidative stress. The latter property of hemin has been examined, using murine L1210 cells and three different oxidant generating systems: (i) glucose/glucose oxidase, (ii) near-ultraviolet irradiation, and (iii) dye-mediated photodynamic action. Cells treated with the lipophilic iron donor ferric-8-hydroxyquinoline, Fe(HQ)2(1 μm, 30 min) were found to be more sensitive to oxidative killing than nontreated controls. However, cells challenged after long-term (20-24 h) exposure to hemin (10 μm) were substantially more resistant than controls and were sensitized far less by Fe(HQ)2. Immunoblot analyses of 24-h hemin-treated cells indicated that the ferritin heavy (H) subunit was elevated 12- to 15-fold, whereas the light (L) subunit was essentially unchanged. Experiments carried out with55Fe(HQ)2showed that iron uptake capacity of cells was greatly enhanced after hemin treatment. More specifically, hemin-stimulated cells were found to contain ∼9 times more immunoprecipitable ferritin iron after incubation with saturating levels (4–5 μm) of55Fe(HQ)2and ∼3 times more iron per ferritin molecule compared with nonstimulated controls. The nonferritin iron content of the latter was estimated to be ∼40 times greater than that of the former following low-level (0.5 μm)55Fe(HQ)2treatment. These results are consistent with the idea that induced ferritin, enriched in H-chain, sequesters redox active iron rapidly and copiously, thereby enhancing cellular resistance to oxidants.