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. 2002 Feb 15;362(Pt 1):137–147. doi: 10.1042/0264-6021:3620137

Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor.

Frank Petrat 1, Daniela Weisheit 1, Martina Lensen 1, Herbert de Groot 1, Reiner Sustmann 1, Ursula Rauen 1
PMCID: PMC1222370  PMID: 11829750

Abstract

Mitochondrial chelatable ("redox-active") iron is considered to contribute to several human diseases, but has not yet been characterized in viable cells. In order to determine this iron pool, we synthesized a new fluorescent indicator, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). In a cell-free system, RPA fluorescence was strongly and stoichiometrically quenched by Fe(2+). RPA selectively accumulated in the mitochondria of cultured rat hepatocytes. The intramitochondrial RPA fluorescence was quenched when iron was added to the cells in a membrane-permeant form. It increased when the mitochondrial chelatable iron available to the probe was experimentally decreased by the membrane-permeant transition metal chelators pyridoxal isonicotinoyl hydrazone and 1,10-phenanthroline. The concentration of mitochondrial chelatable iron in cultured rat hepatocytes, quantified from the increase in RPA fluorescence after addition of pyridoxal isonicotinoyl hydrazone, was found to be 12.2 +/- 4.9 microM. Inhibition of haem synthesis with succinylacetone did not alter the signal obtained in hepatocytes, but a rapid increase in the concentration of mitochondrial chelatable iron was observed in human erythroleukaemia K562 cells. In conclusion, RPA enables the selective determination of the highly physiologically and pathophysiologically interesting mitochondrial pool of chelatable iron in intact cells and to record the time course of alterations of this pool.

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Selected References

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  1. Askwith C., Kaplan J. Iron and copper transport in yeast and its relevance to human disease. Trends Biochem Sci. 1998 Apr;23(4):135–138. doi: 10.1016/s0968-0004(98)01192-x. [DOI] [PubMed] [Google Scholar]
  2. Baliga R., Ueda N., Shah S. V. Increase in bleomycin-detectable iron in ischaemia/reperfusion injury to rat kidneys. Biochem J. 1993 May 1;291(Pt 3):901–905. doi: 10.1042/bj2910901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baliga R., Ueda N., Walker P. D., Shah S. V. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev. 1999 Nov;31(4):971–997. doi: 10.1081/dmr-100101947. [DOI] [PubMed] [Google Scholar]
  4. Balla G., Vercellotti G. M., Eaton J. W., Jacob H. S. Iron loading of endothelial cells augments oxidant damage. J Lab Clin Med. 1990 Oct;116(4):546–554. [PubMed] [Google Scholar]
  5. Beal M. F. Mitochondrial dysfunction in neurodegenerative diseases. Biochim Biophys Acta. 1998 Aug 10;1366(1-2):211–223. doi: 10.1016/s0005-2728(98)00114-5. [DOI] [PubMed] [Google Scholar]
  6. Benzi G., Moretti A. Are reactive oxygen species involved in Alzheimer's disease? Neurobiol Aging. 1995 Jul-Aug;16(4):661–674. doi: 10.1016/0197-4580(95)00066-n. [DOI] [PubMed] [Google Scholar]
  7. Breuer W., Epsztejn S., Cabantchik Z. I. Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II). J Biol Chem. 1995 Oct 13;270(41):24209–24215. doi: 10.1074/jbc.270.41.24209. [DOI] [PubMed] [Google Scholar]
  8. Ceccarelli D., Gallesi D., Giovannini F., Ferrali M., Masini A. Relationship between free iron level and rat liver mitochondrial dysfunction in experimental dietary iron overload. Biochem Biophys Res Commun. 1995 Apr 6;209(1):53–59. doi: 10.1006/bbrc.1995.1469. [DOI] [PubMed] [Google Scholar]
  9. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr. 2000 Feb;71(2):621S–629S. doi: 10.1093/ajcn/71.2.621s. [DOI] [PubMed] [Google Scholar]
  10. Costantini P., Chernyak B. V., Petronilli V., Bernardi P. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J Biol Chem. 1996 Mar 22;271(12):6746–6751. doi: 10.1074/jbc.271.12.6746. [DOI] [PubMed] [Google Scholar]
  11. Delatycki M. B., Williamson R., Forrest S. M. Friedreich ataxia: an overview. J Med Genet. 2000 Jan;37(1):1–8. doi: 10.1136/jmg.37.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ehrenberg B., Montana V., Wei M. D., Wuskell J. P., Loew L. M. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J. 1988 May;53(5):785–794. doi: 10.1016/S0006-3495(88)83158-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Epsztejn S., Kakhlon O., Glickstein H., Breuer W., Cabantchik I. Fluorescence analysis of the labile iron pool of mammalian cells. Anal Biochem. 1997 May 15;248(1):31–40. doi: 10.1006/abio.1997.2126. [DOI] [PubMed] [Google Scholar]
  14. Evans P. J., Halliwell B. Measurement of iron and copper in biological systems: bleomycin and copper-phenanthroline assays. Methods Enzymol. 1994;233:82–92. doi: 10.1016/s0076-6879(94)33010-7. [DOI] [PubMed] [Google Scholar]
  15. Fandrey J., Frede S., Ehleben W., Porwol T., Acker H., Jelkmann W. Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species. Kidney Int. 1997 Feb;51(2):492–496. doi: 10.1038/ki.1997.68. [DOI] [PubMed] [Google Scholar]
  16. Fuchs O. Ferrochelatase, glutathione peroxidase and transferrin receptor mRNA synthesis and levels in mouse erythroleukemia cells. Stem Cells. 1993 May;11 (Suppl 1):13–23. doi: 10.1002/stem.5530110606. [DOI] [PubMed] [Google Scholar]
  17. Harootunian A. T., Kao J. P., Eckert B. K., Tsien R. Y. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J Biol Chem. 1989 Nov 15;264(32):19458–19467. [PubMed] [Google Scholar]
  18. Hershko C., Link G., Pinson A., Peter H. H., Dobbin P., Hider R. C. Iron mobilization from myocardial cells by 3-hydroxypyridin-4-one chelators: studies in rat heart cells in culture. Blood. 1991 May 1;77(9):2049–2053. [PubMed] [Google Scholar]
  19. Huang A. R., Ponka P. A study of the mechanism of action of pyridoxal isonicotinoyl hydrazone at the cellular level using reticulocytes loaded with non-heme 59Fe. Biochim Biophys Acta. 1983 Jun 9;757(3):306–315. doi: 10.1016/0304-4165(83)90056-9. [DOI] [PubMed] [Google Scholar]
  20. Johnson F. B., Sinclair D. A., Guarente L. Molecular biology of aging. Cell. 1999 Jan 22;96(2):291–302. doi: 10.1016/s0092-8674(00)80567-x. [DOI] [PubMed] [Google Scholar]
  21. Nunez M. T., Cole E. S., Glass J. The reticulocyte plasma membrane pathway of iron uptake as determined by the mechanism of alpha, alpha'-dipyridyl inhibition. J Biol Chem. 1983 Jan 25;258(2):1146–1151. [PubMed] [Google Scholar]
  22. Ollinger K., Roberg K. Nutrient deprivation of cultured rat hepatocytes increases the desferrioxamine-available iron pool and augments the sensitivity to hydrogen peroxide. J Biol Chem. 1997 Sep 19;272(38):23707–23711. doi: 10.1074/jbc.272.38.23707. [DOI] [PubMed] [Google Scholar]
  23. Ozawa T., Hayakawa M., Katsumata K., Yoneda M., Ikebe S., Mizuno Y. Fragile mitochondrial DNA: the missing link in the apoptotic neuronal cell death in Parkinson's disease. Biochem Biophys Res Commun. 1997 Jun 9;235(1):158–161. doi: 10.1006/bbrc.1997.6754. [DOI] [PubMed] [Google Scholar]
  24. Petrat F., Rauen U., de Groot H. Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK. Hepatology. 1999 Apr;29(4):1171–1179. doi: 10.1002/hep.510290435. [DOI] [PubMed] [Google Scholar]
  25. Petrat F., de Groot H., Rauen U. Determination of the chelatable iron pool of single intact cells by laser scanning microscopy. Arch Biochem Biophys. 2000 Apr 1;376(1):74–81. doi: 10.1006/abbi.2000.1711. [DOI] [PubMed] [Google Scholar]
  26. Petrat F., de Groot H., Rauen U. Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells. Biochem J. 2001 May 15;356(Pt 1):61–69. doi: 10.1042/0264-6021:3560061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ponka P. Tissue-specific regulation of iron metabolism and heme synthesis: distinct control mechanisms in erythroid cells. Blood. 1997 Jan 1;89(1):1–25. [PubMed] [Google Scholar]
  28. Porter J. B., Gyparaki M., Burke L. C., Huehns E. R., Sarpong P., Saez V., Hider R. C. Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant. Blood. 1988 Nov;72(5):1497–1503. [PubMed] [Google Scholar]
  29. Puccio H., Koenig M. Recent advances in the molecular pathogenesis of Friedreich ataxia. Hum Mol Genet. 2000 Apr 12;9(6):887–892. doi: 10.1093/hmg/9.6.887. [DOI] [PubMed] [Google Scholar]
  30. Rauen U., Petrat F., Li T., De Groot H. Hypothermia injury/cold-induced apoptosis--evidence of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation. FASEB J. 2000 Oct;14(13):1953–1964. doi: 10.1096/fj.00-0071com. [DOI] [PubMed] [Google Scholar]
  31. Schapira A. H. Mitochondrial dysfunction in neurodegenerative disorders. Biochim Biophys Acta. 1998 Aug 10;1366(1-2):225–233. doi: 10.1016/s0005-2728(98)00115-7. [DOI] [PubMed] [Google Scholar]
  32. Sergent O., Morel I., Cogrel P., Chevanne M., Pasdeloup N., Brissot P., Lescoat G., Cillard P., Cillard J. Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures. Relationship with lipid peroxidation. Biol Trace Elem Res. 1995 Jan-Mar;47(1-3):185–192. doi: 10.1007/BF02790116. [DOI] [PubMed] [Google Scholar]
  33. Tangerås A., Flatmark T., Bäckström D., Ehrenberg A. Mitochondrial iron not bound in heme and iron-sulfur centers. Estimation, compartmentation and redox state. Biochim Biophys Acta. 1980 Feb 8;589(2):162–175. doi: 10.1016/0005-2728(80)90035-3. [DOI] [PubMed] [Google Scholar]
  34. Vatassery G. T., Smith W. E., Quach H. T., Lai J. C. In vitro oxidation of vitamin E, vitamin C, thiols and cholesterol in rat brain mitochondria incubated with free radicals. Neurochem Int. 1995 May;26(5):527–535. doi: 10.1016/0197-0186(94)00147-m. [DOI] [PubMed] [Google Scholar]
  35. Voogd A., Sluiter W., van Eijk H. G., Koster J. F. Low molecular weight iron and the oxygen paradox in isolated rat hearts. J Clin Invest. 1992 Nov;90(5):2050–2055. doi: 10.1172/JCI116086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walker P. D., Barri Y., Shah S. V. Oxidant mechanisms in gentamicin nephrotoxicity. Ren Fail. 1999 May-Jul;21(3-4):433–442. doi: 10.3109/08860229909085109. [DOI] [PubMed] [Google Scholar]
  37. de Groot H., Brecht M. Reoxygenation injury in rat hepatocytes: mediation by O2/H2O2 liberated by sources other than xanthine oxidase. Biol Chem Hoppe Seyler. 1991 Jan;372(1):35–41. doi: 10.1515/bchm3.1991.372.1.35. [DOI] [PubMed] [Google Scholar]

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