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Phenanthrolines Protect Astrocytes from Hemin Without Chelating Iron

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Abstract

Hemin, the degradation product of hemoglobin, contributes to the neurodegeneration that occurs in the weeks following a hemorrhagic stroke. The breakdown of hemin in cells releases redox-active iron that can facilitate the production of toxic hydroxyl radicals. The present study used 3-week old primary cultures of mouse astrocytes to compare the toxicity of 33 μM hemin in the presence of the iron chelator 1,10-phenanthroline or its non-chelating analogue, 4,7-phenanthroline. This concentration of hemin killed approximately 75 % of astrocytes within 24 h. Both isoforms of phenanthroline significantly decreased the toxicity of hemin, with the non-chelating analogue providing complete protection at concentrations of 33 μM and above. The decrease in toxicity was associated with less cellular accumulation of hemin. Approximately 90 % of the hemin accumulated was not degraded, irrespective of treatment condition. These observations indicate that chelatable iron is not the cause of hemin toxicity. Cell-free experiments demonstrated that hemin can inactivate a molar excess of hydrogen peroxide (H2O2), and that the rate of inactivation is halved in the presence of either isoform of phenanthroline. We conclude that phenanthrolines may protect astrocytes by limiting hemin uptake and by impairing the capacity of intact hemin to interact with endogenous H2O2.

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References

  1. Andersen KK, Olsen TS, Dehlendorff C, Kammersgaard LP (2009) Hemorrhagic and ischemic strokes compared: stroke severity, mortality, and risk factors. Stroke 40:2068–2072

    Article  PubMed  Google Scholar 

  2. Qureshi AI, Mendelow AD, Hanley DF (2009) Intracerebral haemorrhage. Lancet 373:1632–1644

    Article  PubMed Central  PubMed  Google Scholar 

  3. Dang TN, Bishop GM, Dringen R, Robinson SR (2010) The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin. Glia 58:55–65

    Article  PubMed  Google Scholar 

  4. Kumar S, Bandyopadhyay U (2005) Free heme toxicity and its detoxification systems in human. Toxicol Lett 157:175–188

    Article  PubMed  CAS  Google Scholar 

  5. Balla G, Jacob HS, Eaton JW, Belcher JD, Vercellotti GM (1991) Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury. Arterioscler Thromb 11:1700–1711

    Article  PubMed  CAS  Google Scholar 

  6. Huffman LJ, Miles PR, Shi X, Bowman L (2000) Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages. Exp Lung Res 26:203–217

    Article  PubMed  CAS  Google Scholar 

  7. Nagababu E, Rifkind JM (2004) Heme degradation by reactive oxygen species. Antioxid Redox Signal 6:967–978

    Article  PubMed  CAS  Google Scholar 

  8. Wagner KR, Sharp FR, Ardizzone TD, Lu A, Clark JF (2003) Heme and iron metabolism: role in cerebral hemorrhage. J Cereb Blood Flow Metab 23:629–652

    Article  PubMed  CAS  Google Scholar 

  9. Robinson SR, Dang TN, Dringen R, Bishop GM (2009) Hemin toxicity: a preventable source of brain damage following hemorrhagic stroke. Redox Rep 14:228–235

    Article  PubMed  CAS  Google Scholar 

  10. Halliwell B, Gutteridge J (2007) Free radicals in biology and medicine. Oxford University Press, New York

    Google Scholar 

  11. Gaasch JA, Lockman PR, Geldenhuys WJ, Allen DD, Van Der Schyf CJ (2007) Brain iron toxicity: differential responses of astrocytes, neurons, and endothelial cells. Neurochem Res 32:1196–1208

    Article  PubMed  CAS  Google Scholar 

  12. Moos T, Nielsen TR, Skjørringe T, Morgan EH (2007) Iron trafficking inside the brain. J Neurochem 103:1730–1740

    Article  PubMed  CAS  Google Scholar 

  13. Balla J, Vercellotti GM, Jeney V, Yachie A, Varga Z, Jacob HS, Eaton JW, Balla G (2007) Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal 9:2119–2137

    Article  PubMed  CAS  Google Scholar 

  14. Ke Y, Qian ZM (2007) Brain iron metabolism: neurobiology and neurochemistry. Prog Neurobiol 83:149–173

    Article  PubMed  CAS  Google Scholar 

  15. Li Z, Chen-Roetling J, Regan RF (2009) Increasing expression of H- or L-ferritin protects cortical astrocytes from hemin toxicity. Free Radic Res 43:613–621

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  16. Sigel A, Sigel H (2004) Metal ions and their complexes in medication. Marcel Dekker, New York

    Book  Google Scholar 

  17. Masuda T, Hida H, Kanda Y, Aihara N, Ohta K, Yamada K, Nishino H (2007) Oral administration of metal chelator ameliorates motor dysfunction after a small hemorrhage near the internal capsule in rat. J Neurosci Res 85:213–222

    Article  PubMed  CAS  Google Scholar 

  18. Hoepken HH, Korten T, Robinson SR, Dringen R (2004) Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate. J Neurochem 88:1194–1202

    Article  PubMed  CAS  Google Scholar 

  19. Wu H, Wu T, Xu X, Wang J (2010) Iron toxicity in mice with collagenase-induced intracerebral hemorrhage. J Cereb Blood Flow Metab 31:1243–1250

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  20. Halliwell B (1989) Protection against tissue damage in vivo by desferrioxamine: What is its mechanism of action? Free Radic Biol Med 7:645–651

    Article  PubMed  CAS  Google Scholar 

  21. Regan RF, Rogers B (2003) Delayed treatment of hemoglobin neurotoxicity. J Neurotrauma 20:111–120

    Article  PubMed  Google Scholar 

  22. Vanderveldt GM, Regan RF (2004) The neurotoxic effect of sickle cell hemoglobin. Free Radic Res 38:431–437

    Article  PubMed  CAS  Google Scholar 

  23. Regan RF, Wang Y, Xin M, Chong A, Guo Y (2001) Activation of extracellular signal-regulated kinases potentiates hemin toxicity in astrocyte cultures. J Neurochem 79:545–555

    Article  PubMed  CAS  Google Scholar 

  24. Regan RF, Chen J, Benvenisti-Zarom L (2004) Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin. BMC Neurosci 5:34

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  25. Hamprecht B, Loffler F (1985) Primary glial cultures as a model for studying hormone action. Methods Enzymol 109:341–345

    Article  PubMed  CAS  Google Scholar 

  26. Dringen R, Kussmaul L, Hamprecht B (1998) Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Protoc 2:223–228

    Article  CAS  Google Scholar 

  27. Riemer J, Hoepken HH, Czerwinska H, Robinson SR, Dringen R (2004) Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Anal Biochem 331:370–375

    Article  PubMed  CAS  Google Scholar 

  28. Dang TN, Bishop GM, Dringen R, Robinson SR (2011) The metabolism and toxicity of hemin in astrocytes. Glia 59:1540–1550

    Article  PubMed  Google Scholar 

  29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  30. Liddell JR, Hoepken HH, Crack PJ, Robinson SR, Dringen R (2006) Glutathione peroxidase 1 and glutathione are required to protect mouse astrocytes from iron-mediated hydrogen peroxide toxicity. J Neurosci Res 84:578–586

    Article  PubMed  CAS  Google Scholar 

  31. Liu Y, Ortiz De Montellano PR (2000) Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1. J Biol Chem 275:5297–5307

    Article  PubMed  CAS  Google Scholar 

  32. Laird MD, Wakade C, Alleyne CH Jr, Dhandapani KM (2008) Hemin-induced necroptosis involves glutathione depletion in mouse astrocytes. Free Radic Biol Med 45:1103–1114

    Article  PubMed  CAS  Google Scholar 

  33. Lee J, Yim YS, Ko SJ, Kim DG, Kim CH (2011) Gap junctions contribute to astrocytic resistance against zinc toxicity. Brain Res Bull 86:314–318

    Article  PubMed  CAS  Google Scholar 

  34. Steed JW, Atwood JL (2009) Supramolecular chemistry. Wiley, West Sussex, United Kingdom

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Acknowledgments

We would like to acknowledge the School of Psychology and Psychiatry at Monash University, Melbourne Australia as some data for this project was collected at the Clayton Campus. We would like to thank Hania Czerwinska for her technical assistance with some aspects of this project.

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Authors and Affiliations

  1. School of Health Sciences, RMIT University, PO Box 71, Bundoora, VIC, 3083, Australia

    Jessica E. Owen, Glenda M. Bishop & Stephen R. Robinson

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  1. Jessica E. Owen
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  2. Glenda M. Bishop
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  3. Stephen R. Robinson
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Correspondence to Stephen R. Robinson.

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Owen, J.E., Bishop, G.M. & Robinson, S.R. Phenanthrolines Protect Astrocytes from Hemin Without Chelating Iron. Neurochem Res 39, 693–699 (2014). https://doi.org/10.1007/s11064-014-1256-8

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  • DOI: https://doi.org/10.1007/s11064-014-1256-8

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