title: The role of endocytic pathways in cellular uptake of plasma non-transferrin iron.
abstract: BACKGROUND
  In transfusional siderosis, the iron binding capacity of plasma transferrin is often surpassed, with concomitant generation of non-transferrin-bound iron. Although implicated in tissue siderosis, non-transferrin-bound iron modes of cell ingress remain undefined, largely because of its variable composition and association with macromolecules. Using fluorescent tracing of labile iron in endosomal vesicles and cytosol, we examined the hypothesis that non-transferrin-bound iron fractions detected in iron overloaded patients enter cells via bulk endocytosis.
  
  
  METHODS
  Fluorescence microscopy and flow cytometry served as analytical tools for tracing non-transferrin-bound iron entry into endosomes with the redox-reactive macromolecular probe Oxyburst-Green and into the cytosol with cell-laden calcein green and calcein blue. Non-transferrin-bound iron-containing media were from sera of polytransfused thalassemia major patients and model iron substances detected in thalassemia major sera; cell models were cultured macrophages, and cardiac myoblasts and myocytes.
  
  
  RESULTS
  Exposure of cells to ferric citrate together with albumin, or to non-transferrin-bound iron-containing sera from thalassemia major patients caused an increase in labile iron content of endosomes and cytosol in macrophages and cardiac cells. This increase was more striking in macrophages, but in both cell types was largely reduced by co-exposure to non-transferrin-bound iron-containing media with non-penetrating iron chelators or apo-transferrin, or by treatment with inhibitors of endocytosis. Endosomal iron accumulation traced with calcein-green was proportional to input non-transferrin-bound iron levels (r(2) = 0.61) and also preventable by pre-chelation.
  
  
  CONCLUSIONS
  Our studies indicate that macromolecule-associated non-transferrin-bound iron can initially gain access into various cells via endocytic pathways, followed by iron translocation to the cytosol. Endocytic uptake of plasma non-transferrin-bound iron is a possible mechanism that can contribute to iron loading of cell types engaged in bulk/adsorptive endocytosis, highlighting the importance of its prevention by iron chelation.
authors.affiliation: The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Safra Campus at Givat Ram, Jerusalem 91904, Israel.
authors.forename: Yang-Sung
authors.initials: YS
authors.lastname: Sohn
authors.forename: Hussam
authors.initials: H
authors.lastname: Ghoti
authors.forename: William
authors.initials: W
authors.lastname: Breuer
authors.forename: Eliezer
authors.initials: E
authors.lastname: Rachmilewitz
authors.forename: Samah
authors.initials: S
authors.lastname: Attar
authors.forename: Guenter
authors.initials: G
authors.lastname: Weiss
authors.forename: Z Ioav
authors.initials: ZI
authors.lastname: Cabantchik
mesh_terms: D000293:Adolescent; D000328:Adult; D001692:Biological Transport; D015415:Biomarkers / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology; D007501:Iron / Q000097:blood / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism*; D055815:Young Adult; D017086:beta-Thalassemia / Q000378:metabolism* / Q000473:pathology
chemical_list: D015415:Biomarkers; D007502:Iron Chelating Agents; D014168:Transferrin; D007501:Iron