Chelation and Mobilization of Cellular Iron by Different Classes of Chelators

  1. G. Zanninelli1,2,
  2. H. Glickstein1,
  3. W. Breuer1,
  4. P. Milgram1,
  5. P. Brissot2,
  6. R. C. Hider3,
  7. A. M. Konijn4,
  8. J. Libman5,
  9. A. Shanzer5 and
  10. Z. Ioav Cabantchik1
  1. 1Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel 91904 (G.Z., H.G., W.B., P.M., Z.I.C.), 2Liver Research Unit, Institut National de la Santé et de la Recherche Medicale U-49, Pontchaillou University Hospital, 35033 Rennes, France (G.Z., P.B.), 3Department of Pharmacy, King’s College, W8 7AH London, UK (R.C.H.), 4Department of Public Health, Hadassah Medical School, Jerusalem, 91120 Israel (A.M.K.), and5Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel (J.L., A.S.)

    Abstract

    Iron chelators belonging to three distinct chemical families were assessed in terms of their physicochemical properties and the kinetics of iron chelation in solution and in two biological systems. Several hydroxypyridinones, reversed siderophores, and desferrioxamine derivatives were selected to cover agents with different iron-binding stoichiometry and geometry and a wide range of lipophilicity, as determined by the octanol-water partition coefficients. The selection also included highly lipophilic chelators with potentially cell-cleavable ester groups that can serve as precursors of hydrophilic and membrane-impermeant chelators. Iron binding was determined by the chelator capacity for restoring the fluorescence of iron-quenched calcein (CA), a dynamic fluorescent metallosensor. The iron-scavenging properties of the chelators were assessed under three different conditions: (a) in solution, by mixing iron salts with free CA; (b) in resealed red cell ghosts, by encapsulation of CA followed by loading with iron; and (c) in human erythroleukemia K562 cells, by loading with the permeant CA-acetomethoxy ester, in situ formation of free CA, and binding of cytosolic labile iron. The time-dependent recovery of fluorescence in the presence of a given chelator provided a continuous measure for the capacity of the chelator to access the iron/CA-containing compartment. The resulting rate constants of fluorescence recovery indicated that chelation in solution was comparable for the members of each family of chelators, whereas chelation in either biological system was largely dictated by the lipophilicity of the free chelator. For example, desferrioxamine was among the fastest and most efficient iron scavengers in solution but was essentially ineffective in either biological system when used at ≤200 μm over a 2-hr period at 37°. On the other hand, the highly lipophilic and potentially cell-cleavable hydroxypyridinones and reversed siderophores were highly efficient in all biological systems tested. It is implied that in K562 cells, hydrolysis of these chelators is relatively slower than their ingress and binding of intracellular iron. The chelator-mediated translocation of iron from cells to medium was assessed in 55Fe-transferrin-loaded K562 cells. The speed of iron mobilization by members of the three families of chelators correlated with the lipophilicity of the free ligand or the iron-complexed chelator. The acquired information is of relevance for the design of chelators with improved biological performance.

    Footnotes

    • Send reprint requests to: Dr. Z. Ioav Cabantchik, Department of Biological Chemistry, Institute of Life Sciences, Hebrew University Jerusalem, Jerusalem, Israel 91904. E-mail:ioav{at}cc.huji.ac.il

    • ↵1 A. M. Albrecht-Gary, personal communication.

    • ↵2 It remains to be determined whether the esterified HPO117 is cleaved inside K562 cells to its free acid, HPO102. In rats, the metabolite HPO102 was indeed detected in the bile 10 min after intravenous administration of HPO117 (17).

    • ↵3 G. Zanninelli and Z. Ioav Cabantchik, unpublished observations.

    • This work was supported in part by Israel National Science Fund administered by the Israeli Academy of Sciences (Z.I.C.), European Commission Grant BMH4-97-2149 (Z.I.C., P.B., R.H), Israel Ministry of Health (Z.I.C.), and Arc en Ciel Program for French-Israel cooperation (Z.I.C., P.B.). G.Z. was a recipient of a short term fellowship from the European Science Foundation.

    • This work is dedicated to the memory of J.L., who passed away in March 1997.

    • Abbreviations:
      DFO
      desferrioxamine
      HPO
      hydroxypyridinone
      RSF
      reversed siderophore
      CA
      calcein
      CA-AM
      calcein-acetomethoxy ester
      Tf
      transferrin
      PC
      partition coefficient
      LIP
      labile iron pool
      FAS
      ferrous ammonium sulfate
      SIH
      salicylaldehyde isonicotinoyl hydrazone
      HBS
      HEPES-buffered saline
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      MEM
      minimal essential medium
      α-MEM-HEPES
      HEPES-buffered, bicarbonate-free α-MEM containing 20 mm HEPES, pH 7.3
      DMSO
      dimethylsulfoxide
      • Received April 9, 1996.
      • Accepted January 8, 1997.
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    1. Molecular Pharmacology May 1, 1997 vol. 51 no. 5 842-852
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