Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 2001 May 15;356(Pt 1):61–69. doi: 10.1042/0264-6021:3560061

Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells.

F Petrat 1, H de Groot 1, U Rauen 1
PMCID: PMC1221812  PMID: 11336636

Abstract

The pool of cellular chelatable iron ('free iron', 'low-molecular-weight iron', the 'labile iron pool') is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8+/-2.6 microM within the cytosol and of at least 4.8 microM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6+/-2.9 microM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3+/-2.6 microM; nucleus, 11.8+/-3.9 microM; mitochondria, 9.2+/-2.7 microM); in addition, chelatable iron (approx. 16+/-4 microM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

Full Text

The Full Text of this article is available as a PDF (278.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. 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]
  4. Blouin A., Bolender R. P., Weibel E. R. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol. 1977 Feb;72(2):441–455. doi: 10.1083/jcb.72.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cai C. X., Birk D. E., Linsenmayer T. F. Nuclear ferritin protects DNA from UV damage in corneal epithelial cells. Mol Biol Cell. 1998 May;9(5):1037–1051. doi: 10.1091/mbc.9.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cairo G., Tacchini L., Pogliaghi G., Anzon E., Tomasi A., Bernelli-Zazzera A. Induction of ferritin synthesis by oxidative stress. Transcriptional and post-transcriptional regulation by expansion of the "free" iron pool. J Biol Chem. 1995 Jan 13;270(2):700–703. doi: 10.1074/jbc.270.2.700. [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. Csermely P., Fodor P., Somogyi J. The tumor promoter tetradecanoylphorbol-13-acetate elicits the redistribution of heavy metals in subcellular fractions of rabbit thymocytes as measured by plasma emission spectroscopy. Carcinogenesis. 1987 Nov;8(11):1663–1666. doi: 10.1093/carcin/8.11.1663. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Foury F., Cazzalini O. Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria. FEBS Lett. 1997 Jul 14;411(2-3):373–377. doi: 10.1016/s0014-5793(97)00734-5. [DOI] [PubMed] [Google Scholar]
  12. Gurgueira S. A., Meneghini R. An ATP-dependent iron transport system in isolated rat liver nuclei. J Biol Chem. 1996 Jun 7;271(23):13616–13620. doi: 10.1074/jbc.271.23.13616. [DOI] [PubMed] [Google Scholar]
  13. Halliwell B., Gutteridge J. M. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1–85. doi: 10.1016/0076-6879(90)86093-b. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Lai C. C., Huang W. H., Klevay L. M., Gunning W. T., 3rd, Chiu T. H. Antioxidant enzyme gene transcription in copper-deficient rat liver. Free Radic Biol Med. 1996;21(2):233–240. doi: 10.1016/0891-5849(96)00029-9. [DOI] [PubMed] [Google Scholar]
  17. Lytton S. D., Mester B., Dayan I., Glickstein H., Libman J., Shanzer A., Cabantchik Z. I. Mode of action of iron (III) chelators as antimalarials: I. Membrane permeation properties and cytotoxic activity. Blood. 1993 Jan 1;81(1):214–221. [PubMed] [Google Scholar]
  18. Meneghini R. Iron homeostasis, oxidative stress, and DNA damage. Free Radic Biol Med. 1997;23(5):783–792. doi: 10.1016/s0891-5849(97)00016-6. [DOI] [PubMed] [Google Scholar]
  19. Myers B. M., Tietz P. S., Tarara J. E., LaRusso N. F. Dynamic measurements of the acute and chronic effects of lysosomotropic agents on hepatocyte lysosomal pH using flow cytometry. Hepatology. 1995 Nov;22(5):1519–1526. [PubMed] [Google Scholar]
  20. Nankivell B. J., Tay Y. C., Boadle R. A., Harris D. C. Dietary protein alters tubular iron accumulation after partial nephrectomy. Kidney Int. 1994 Apr;45(4):1006–1013. doi: 10.1038/ki.1994.136. [DOI] [PubMed] [Google Scholar]
  21. Nicotera P., Zhivotovsky B., Orrenius S. Nuclear calcium transport and the role of calcium in apoptosis. Cell Calcium. 1994 Oct;16(4):279–288. doi: 10.1016/0143-4160(94)90091-4. [DOI] [PubMed] [Google Scholar]
  22. Nielsen P., Düllmann J., Wulfhekel U., Heinrich H. C. Non-transferrin-bound-iron in serum and low-molecular-weight-iron in the liver of dietary iron-loaded rats. Int J Biochem. 1993 Feb;25(2):223–232. doi: 10.1016/0020-711x(93)90010-c. [DOI] [PubMed] [Google Scholar]
  23. Nilsson E., Ghassemifar R., Brunk U. T. Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress. Histochem J. 1997 Nov-Dec;29(11-12):857–865. doi: 10.1023/a:1026441907803. [DOI] [PubMed] [Google Scholar]
  24. Ollinger K., Brunk U. T. Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radic Biol Med. 1995 Nov;19(5):565–574. doi: 10.1016/0891-5849(95)00062-3. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Pastorino J. G., Snyder J. W., Hoek J. B., Farber J. L. Ca2+ depletion prevents anoxic death of hepatocytes by inhibiting mitochondrial permeability transition. Am J Physiol. 1995 Mar;268(3 Pt 1):C676–C685. doi: 10.1152/ajpcell.1995.268.3.C676. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Rauen U., Hanssen M., Lauchart W., Becker H. D., de Groot H. Energy-dependent injury to cultured sinusoidal endothelial cells of the rat liver in UW solution. Transplantation. 1993 Mar;55(3):469–473. doi: 10.1097/00007890-199303000-00002. [DOI] [PubMed] [Google Scholar]
  32. Rauen U., Polzar B., Stephan H., Mannherz H. G., de Groot H. Cold-induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species. FASEB J. 1999 Jan;13(1):155–168. doi: 10.1096/fasebj.13.1.155. [DOI] [PubMed] [Google Scholar]
  33. Recalcati S., Conte D., Cairo G. Preferential activation of iron regulatory protein-2 in cell lines as a result of higher sensitivity to iron. Eur J Biochem. 1999 Jan;259(1-2):304–309. doi: 10.1046/j.1432-1327.1999.00038.x. [DOI] [PubMed] [Google Scholar]
  34. Smith A. G., Carthew P., Francis J. E., Edwards R. E., Dinsdale D. Characterization and accumulation of ferritin in hepatocyte nuclei of mice with iron overload. Hepatology. 1990 Dec;12(6):1399–1405. doi: 10.1002/hep.1840120622. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Taylor D. E., Ghio A. J., Piantadosi C. A. Reactive oxygen species produced by liver mitochondria of rats in sepsis. Arch Biochem Biophys. 1995 Jan 10;316(1):70–76. doi: 10.1006/abbi.1995.1011. [DOI] [PubMed] [Google Scholar]
  37. Ueda N., Guidet B., Shah S. V. Gentamicin-induced mobilization of iron from renal cortical mitochondria. Am J Physiol. 1993 Sep;265(3 Pt 2):F435–F439. doi: 10.1152/ajprenal.1993.265.3.F435. [DOI] [PubMed] [Google Scholar]
  38. Zahrebelski G., Nieminen A. L., al-Ghoul K., Qian T., Herman B., Lemasters J. J. Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study. Hepatology. 1995 May;21(5):1361–1372. [PubMed] [Google Scholar]
  39. 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]
  40. von Zglinicki T., Brunk U. T. Intracellular interactions under oxidative stress and aging: a hypothesis. Z Gerontol. 1993 Jul-Aug;26(4):215–220. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES