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Molecular and Cellular Biochemistry

, Volume 413, Issue 1–2, pp 199–215 | Cite as

Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells

  • Tareck Rharass
  • Adam Gbankoto
  • Christophe Canal
  • Gizem Kurşunluoğlu
  • Amandine Bijoux
  • Daniela Panáková
  • Anne-Cécile Ribou
Article

Abstract

The implication of oxidative stress as primary mechanism inducing doxorubicin (DOX) cardiotoxicity is still questionable as many in vitro studies implied supra-clinical drug doses or unreliable methodologies for reactive oxygen species (ROS) detection. The aim of this study was to clarify whether oxidative stress is involved in compliance with the conditions of clinical use of DOX, and using reliable tools for ROS detection. We examined the cytotoxic mechanisms of 2 μM DOX 1 day after the beginning of the treatment in differentiated H9c2 rat embryonic cardiac cells. Cells were exposed for 2 or 24 h with DOX to mimic a single chronic dosage or to favor accumulation, respectively. We found that apoptosis was prevalent in cells exposed for a short period with DOX: cells showed typical hallmarks as loss of anchorage ability, mitochondrial hyperpolarization followed by the collapse of mitochondrial activity, and nuclear condensation. Increasing the exposure period favored a shift to necrosis as the cells preferentially exhibited early DNA impairment and nuclear swelling. In either case, measuring the fluorescence lifetime of 1-pyrenebutyric acid or the intensities of dihydroethidium or amplex red showed a consistent pattern in ROS production which was a slight increased level far from representative of an oxidative stress. Moreover, pre-treatment with dexrazoxane provided a cytoprotective effect although it failed to detoxify ROS. Our data support that oxidative stress is unlikely to be the primary mechanism of DOX cardiac toxicity in vitro.

Keywords

Apoptosis Cardiotoxicity Dexrazoxane Doxorubicin Necrosis Oxidative stress 

Notes

Acknowledgments

This paper is dedicated to the memory of our friend and colleague Jean Vigo who passed away in July 2015. His enthusiasm and tireless activity on improving instruments for fluorescence lifetime detection will remain a model. We would like to thank Christoph Grunau for proof reading the article. This work was supported by funds from the French “Ligue Nationale Contre le Cancer” (Comités des Pyrénées-Orientales et du Gard) to T.R., A.G., C.C., A.B., J.V. and A.C.R., the ERASMUS program to G.K., and by Helmholtz Young Investigator Program to T.R. and D.P.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11010_2016_2653_MOESM1_ESM.pdf (685 kb)
Supplementary material 1 (PDF 684 kb)
11010_2016_2653_MOESM2_ESM.pdf (4 kb)
Supplementary material 2 (PDF 4 kb)

References

  1. 1.
    Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2010) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56:185–229CrossRefGoogle Scholar
  2. 2.
    Pommier Y, Leo E, Zhang H, Marchand C (2010) DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol 17:421–433CrossRefPubMedGoogle Scholar
  3. 3.
    Cutts SM, Nudelman A, Rephaeli A, Phillips DR (2005) The power and potential of doxorubicin-DNA adducts. IUBMB Life 57:73–81CrossRefPubMedGoogle Scholar
  4. 4.
    Jung K, Reszka R (2001) Mitochondria as subcellular targets for clinically useful anthracyclines. Adv Drug Deliv Rev 49:87–105CrossRefPubMedGoogle Scholar
  5. 5.
    Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns H, Moens AL (2012) Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol 52:1213–1225CrossRefPubMedGoogle Scholar
  6. 6.
    Štěrba M, Popelová O, Vávrová A, Jirkovský E, Kovaříková P, Geršl V, Šimůnek T (2013) Oxidative stress, redox signaling, and metal chelation in anthracycline cardiotoxicity and pharmacological cardioprotection. Antioxid Redox Signal 18:899–929PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Alberts DS, Hess LM, Von Hoff DD, Dorr RT (2009) Pharmacology and therapeutics in gynecologic cancer. In: Barakat RR, Markman M, Randall ME (eds) Principles and practice of gynecologic oncology, 5th edn. Lippincott Williams and Wilkins, Philadelphia, pp 409–462Google Scholar
  8. 8.
    Kluza J, Marchetti P, Gallego MA, Lancel S, Fournier C, Loyens A, Beauvillain JC, Bailly C (2004) Mitochondrial proliferation during apoptosis induced by anticancer agents: effects of doxorubicin and mitoxantrone on cancer and cardiac cells. Oncogene 23:7018–7030CrossRefPubMedGoogle Scholar
  9. 9.
    Spallarossa P, Garibaldi S, Altieri P, Fabbi P, Manca V, Nasti S, Rossettin P, Ghigliotti G, Ballestrero A, Patrone F, Barsotti A, Brunelli C (2004) Carvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro. J Mol Cell Cardiol 37:837–846CrossRefPubMedGoogle Scholar
  10. 10.
    Tan X, Wang DB, Lu X, Wei H, Zhu R, Zhu SS, Jiang H, Yang ZJ (2010) Doxorubicin induces apoptosis in H9c2 cardiomyocytes: role of overexpressed eukaryotic translation initiation factor 5A. Biol Pharm Bull 33:1666–1672CrossRefPubMedGoogle Scholar
  11. 11.
    Tokarska-Schlattner M, Zaugg M, Zuppinger C, Wallimann T, Schlattner U (2006) New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics. J Mol Cell Cardiol 41:389–405CrossRefPubMedGoogle Scholar
  12. 12.
    Ma J, Wang Y, Zheng D, Wei M, Xu H, Peng T (2013) Rac1 signalling mediates doxorubicin-induced cardiotoxicity through both reactive oxygen species-dependent and -independent pathways. Cardiovasc Res 97:77–87CrossRefPubMedGoogle Scholar
  13. 13.
    Lipshultz SE, Sambatakos P, Maguire M, Karnik R, Ross SW, Franco VI, Miller TL (2014) Cardiotoxicity and cardioprotection in childhood cancer. Acta Haemathol 132:391–399CrossRefGoogle Scholar
  14. 14.
    Galetta F, Franzoni F, Cervetti G, Regoli F, Fallahi P, Tocchini L, Carpi A, Antonelli A, Petrini M, Santoro G (2010) In vitro and in vivo study on the antioxidant activity of dexrazoxane. Biomed Pharmacother 64:259–263CrossRefPubMedGoogle Scholar
  15. 15.
    Junjing Z, Yan Z, Baolu Z (2010) Scavenging effects of dexrazoxane on free radicals. J Clin Biochem Nutr 47:238–245PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Green PS, Leeuwenburgh C (2002) Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. BBA Mol Basis Dis 1588:94–101CrossRefGoogle Scholar
  17. 17.
    Choi EH, Chang HJ, Cho JY, Chun HS (2007) Cytoprotective effect of anthocyanins against doxorubicin-induced toxicity in H9c2 cardiomyocytes in relation to their antioxidant activities. Food Chem Toxicol 45:1873–1881CrossRefPubMedGoogle Scholar
  18. 18.
    Bernuzzi F, Recalcati S, Alberghini A, Cairo G (2009) Reactive oxygen species-independent apoptosis in doxorubicin-treated H9c2 cardiomyocytes: role for heme oxygenase-1 down-modulation. Chem-Biol Interact 177:12–20CrossRefPubMedGoogle Scholar
  19. 19.
    Gilleron M, Marechal X, Montaigne D, Franczak J, Neviere R, Lancel S (2009) NADPH oxidases participate to doxorubicin-induced cardiac myocyte apoptosis. Biochem Biophys Res Commun 388:727–731CrossRefPubMedGoogle Scholar
  20. 20.
    Pereira SL, Ramalho-Santos J, Branco AF, Sardão VM, Oliveira PJ, Carvalho RA (2011) Metabolic remodeling during H9c2 myoblast differentiation: relevance for in vitro toxicity studies. Cardiovasc Toxicol 11:180–190CrossRefPubMedGoogle Scholar
  21. 21.
    Branco AF, Sampaio SF, Moreira AC, Holy J, Wallace KB, Baldeiras I, Oliveira PJ, Sardão VA (2012) Differentiation-dependent doxorubicin toxicity on H9c2 cardiomyoblasts. Cardiovasc Toxicol 12:326–340CrossRefPubMedGoogle Scholar
  22. 22.
    Hasinoff BB, Schnabl KL, Marusak RA, Patel D, Huebner E (2003) Dexrazoxane (ICRF-187) protects cardiac myocytes against doxorubicin by preventing damage to mitochondria. Cardiovasc Toxicol 3:89–99CrossRefPubMedGoogle Scholar
  23. 23.
    Corna G, Santambrogio P, Minotti G, Cairo G (2004) Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity: role of reactive oxygen species and ferritin. J Biol Chem 279:13738–13745CrossRefPubMedGoogle Scholar
  24. 24.
    Ludke A, Sharma AK, Bagchi AK, Singal PK (2012) Subcellular basis of vitamin C protection against doxorubicin-induced changes in rat cardiomyocytes. Mol Cell Biochem 360:215–224CrossRefPubMedGoogle Scholar
  25. 25.
    Gomes A, Fernandes E, Lima JLFC (2005) Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 65:45–80CrossRefPubMedGoogle Scholar
  26. 26.
    Bartosz G (2006) Use of spectroscopic probes for detection of reactive oxygen species. Clin Chim Acta 368:53–76CrossRefPubMedGoogle Scholar
  27. 27.
    Gao J, Yang G, Pi R, Li R, Wang P, Zhang H, Le K, Chen S, Liu P (2008) Tanshinone IIA protects neonatal rat cardiomyocytes from adriamycin-induced apoptosis. Transl Res 151:79–87CrossRefPubMedGoogle Scholar
  28. 28.
    Kalyanaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ, Ischiropoulos H (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 52:1–6PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Shan PR, Xu WW, Huang ZQ, Pu J, Huang WJ (2014) Protective role of retinoid X receptor in H9c2 cardiomyocytes from hypoxia/reoxygenation injury in rats. World J Emerg Med 5:122–127PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Rharass T, Vigo J, Salmon JM, Ribou AC (2006) Variation of 1-pyrenebutyric acid fluorescence lifetime in single living cells treated with molecules increasing or decreasing reactive oxygen species levels. Anal Biochem 357:1–8CrossRefPubMedGoogle Scholar
  31. 31.
    Moné Y, Ribou AC, Cosseau C, Duval D, Théron A, Mitta G, Gourbal B (2011) An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions. Int J Parasitol 41:721–730CrossRefPubMedGoogle Scholar
  32. 32.
    Ribou AC, Reinhardt K (2012) Reduced metabolic rate and oxygen radicals production in stored insect sperm. Proc R Soc B Biol Sci 279:2196–2203CrossRefGoogle Scholar
  33. 33.
    Savatier J, Rharass T, Canal C, Gbankoto A, Vigo J, Salmon JM, Ribou AC (2012) Adriamycin dose and time effects on cell cycle, cell death, and reactive oxygen species generation in leukaemia cells. Leukemia Res 36:791–798CrossRefGoogle Scholar
  34. 34.
    Imondi AR (1998) Preclinical models of cardiac protection and testing for effects of dexrazoxane on doxorubicin antitumor effects. Semin Oncol 25:22–30PubMedGoogle Scholar
  35. 35.
    Schroeder PE, Jensen PB, Sehested M, Hofland KF, Langer SW, Hasinoff BB (2003) Metabolism of dexrazoxane (ICRF-187) used as a rescue agent in cancer patients treated with high-dose etoposide. Cancer Chemother Pharmacol 52:167–174CrossRefPubMedGoogle Scholar
  36. 36.
    Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssière JL, Petit PX, Kroemer G (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181:1661–1672CrossRefPubMedGoogle Scholar
  37. 37.
    Zhivotosky B, Orrenius S (2001) Assessment of apoptosis and necrosis by DNA fragmentation and morphological criteria. Curr Protoc Cell Biol 12:18.3:18.3.1–18.3.23Google Scholar
  38. 38.
    Napirei M, Wulf S, Mannherz HG (2004) Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system. Arthritis Rheum US 50:1873–1883CrossRefGoogle Scholar
  39. 39.
    Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:96–99CrossRefPubMedGoogle Scholar
  40. 40.
    Toné S, Sugimoto K, Tanda K, Suda T, Uehira K, Kanouchi H, Samejima K, Minatogawa Y, Earnshaw WC (2007) Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis. Exp Cell Res 313:3635–3644PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Perl A, Gergely PJ, Nagy G, Koncz A, Banki K (2004) Mitochondrial hyperpolarization: a checkpoint of T-cell life, death and autoimmunity. Trends Immunol 25:360–367PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Zhao H, Joseph J, Fales HM, Sokoloski EA, Levine RL, Vásquez-Vivar J, Kalyanaraman B (2005) Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc Natl Acad Sci USA 102:5727–5732PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Zhao B, Summers FA, Mason RP (2012) Photooxidation of amplex red to resorufin: implications of exposing the amplex red assay to light. Free Radic Biol Med 53:1080–1087PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Grossmann J, Walther K, Artinger M, Kiessling S, Schölmerich J (2001) Apoptotic signaling during initiation of detachment-induced apoptosis (“anoikis”) of primary human intestinal epithelial cells. Cell Growth Differ 12:147–155PubMedGoogle Scholar
  45. 45.
    McCarthy NJ, Evan GI (1997) Methods for detecting and quantifying apoptosis. In: de Pablo F, Ferrús A, Stern CD (eds) Cellular and molecular procedures in developmental biology, vol 36., Current topics in developmental biologyAcademic Press, San Diego, pp 259–278Google Scholar
  46. 46.
    Hirsch T, Marchetti P, Susin SA, Dallaporta B, Zamzami N, Marzo I, Geuskens M, Kroemer G (1997) The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15:1573–1581CrossRefPubMedGoogle Scholar
  47. 47.
    Lemasters JJ (1999) Mechanisms of hepatic toxicity. V. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. Am J Physiol 276:G1–G6PubMedGoogle Scholar
  48. 48.
    Yang F, Teves SS, Kemp CJ, Henikoff S (2014) Doxorubicin, DNA torsion, and chromatin dynamics. BBA Rev Cancer 1845:84–89Google Scholar
  49. 49.
    Bell EL, Klimova TA, Eisenbart J, Moraes CT, Murphy MP, Budinger GR, Chandel NS (2007) The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. J Cell Biol 177:1029–1036PubMedCentralCrossRefPubMedGoogle Scholar
  50. 50.
    Gülden M, Jess A, Kammann J, Maser E, Seibert H (2010) Cytotoxic potency of H2O2 in cell cultures: impact of cell concentration and exposure time. Free Radic Biol Med 49:1298–1305CrossRefPubMedGoogle Scholar
  51. 51.
    Antunes F, Cadenas E (2000) Estimation of H2O2 gradients across biomembranes. FEBS Lett 475:121–126CrossRefPubMedGoogle Scholar
  52. 52.
    Antunes F, Cadenas E (2001) Cellular titration of apoptosis with steady state concentrations of H2O2: submicromolar levels of H2O2 induce apoptosis through fenton chemistry independent of the cellular thiol state. Free Radic Biol Med 30:1008–1018CrossRefPubMedGoogle Scholar
  53. 53.
    Horwitz LD, Leff JA (1995) Catalase and hydrogen peroxide cytotoxicity in cultured cardiac myocytes. J Mol Cell Cardiol 27:909–915CrossRefPubMedGoogle Scholar
  54. 54.
    Kaiserová H, den Hartog GJM, Šimůnek T, Schröterová L, Kvasničková E, Bast A (2006) Iron is not involved in oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin. Br J Pharmacol 149:920–930PubMedCentralCrossRefPubMedGoogle Scholar
  55. 55.
    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Majno G, Joris I (1995) Apoptosis, oncosis and necrosis. An overview of cell death. Am J Pathol 146:3–15PubMedCentralPubMedGoogle Scholar
  57. 57.
    Zeiss CJ (2003) The apoptosis-necrosis continuum: insights from genetically altered mice. Vet Pathol 40:481–495CrossRefPubMedGoogle Scholar
  58. 58.
    Arends MJ, McGregor AH, Wyllie AH (1994) Apoptosis is inversely related to necrosis and determines net growth in tumors bearing constitutively expressed myc, ras, and HPV oncogenes. Am J Pathol 144:1045–1057PubMedCentralPubMedGoogle Scholar
  59. 59.
    Hasinoff BB, Schroeder PE, Patel D (2003) The metabolites of the cardioprotective drug dexrazoxane do not protect myocytes from doxorubicin-induced cytotoxicity. Mol Pharmacol 64:670–678CrossRefPubMedGoogle Scholar
  60. 60.
    Hašková P, Koubková L, Vávrová A, Macková E, Hrušková K, Kovaříková P, Vávrová K, Šimůnek T (2011) Comparison of various iron chelators used in clinical practice as protecting agents against catecholamine-induced oxidative injury and cardiotoxicity. Toxicology 289:122–131CrossRefPubMedGoogle Scholar
  61. 61.
    Lebrecht D, Kokkori A, Ketelsen UP, Setzer B, Walker UA (2005) Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin. J Pathol 207:436–444CrossRefPubMedGoogle Scholar
  62. 62.
    Ito H, Miller SC, Billingham ME, Akimoto H, Torti SV, Wade R, Gahlmann R, Lyons G, Kedes L, Torti FM (1990) Doxorubicin selectively inhibits muscle gene expression in cardiac muscle cells in vivo and in vitro. Proc Natl Acad Sci USA 87:4275–4279PubMedCentralCrossRefPubMedGoogle Scholar
  63. 63.
    Dudnakova TV, Lakomkin VL, Tsyplenkova VG, Shekhonin BV, Shirinsky VP, Kapelko VI (2003) Alterations in myocardial cytoskeletal and regulatory protein expression following a single doxorubicin injection. J Cardiovasc Pharmacol 41:788–794CrossRefPubMedGoogle Scholar
  64. 64.
    Horie T, Ono K, Nishi H, Nagao K, Kinoshita M, Watanabe S, Kuwabara Y, Nakashima Y, Takanabe-Mori R, Nishi E, Hasegawa K, Kita T, Kimura T (2010) Acute doxorubicin cardiotoxicity is associated with miR-146a-induced inhibition of the neuregulin-ErbB pathway. Cardiovasc Res 87:656–664PubMedCentralCrossRefPubMedGoogle Scholar
  65. 65.
    Classen S, Olland S, Berger JM (2003) Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187. Proc Natl Acad Sci U S A 100:10629–10634PubMedCentralCrossRefPubMedGoogle Scholar
  66. 66.
    Lyu YL, Kerrigan JE, Lin CP, Azarova AM, Tsai YC, Ban Y, Liu LF (2007) Topoisomerase IIβ-mediated DNA double-strand breaks: implications in doxorubicin cardiotoxicity and prevention by dexrazoxane. Cancer Res 67:8839–8846CrossRefPubMedGoogle Scholar
  67. 67.
    Martin E, Thougaard AV, Grauslund M, Jensen PB, Bjorkling F, Hasinoff BB, Tjørnelund J, Sehested M, Jensen LH (2009) Evaluation of the topoisomerase II-inactive bisdioxopiperazine ICRF-161 as a protectant against doxorubicin-induced cardiomyopathy. Toxicology 255:72–79CrossRefPubMedGoogle Scholar
  68. 68.
    Deng S, Yan T, Jendrny C, Nemecek A, Vincetic M, Gödtel-Armbrust U, Wojnowski L (2014) Dexrazoxane may prevent doxorubicin-induced DNA damage via depleting both topoisomerase II isoforms. BMC Cancer 14:842PubMedCentralCrossRefPubMedGoogle Scholar
  69. 69.
    Lai R, Long Y, Li Q, Zhang X, Rong T (2011) Oxidative stress markers may not be early markers of doxorubicin-induced cardiotoxicity in rabbits. Exp Ther Med 2:947–950PubMedCentralPubMedGoogle Scholar
  70. 70.
    Zhou S, Palmeira CM, Wallace KB (2001) Doxorubicin-induced persistent oxidative stress to cardiac myocytes. Toxicol Lett 121:151–157CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Tareck Rharass
    • 1
    • 2
  • Adam Gbankoto
    • 3
  • Christophe Canal
    • 1
  • Gizem Kurşunluoğlu
    • 4
  • Amandine Bijoux
    • 1
  • Daniela Panáková
    • 2
  • Anne-Cécile Ribou
    • 1
    • 5
  1. 1.Institute of Modeling and Analysis in Geo-Environmental and Health (IMAGES_ESPACE-DEV)University of Perpignan Via DomitiaPerpignanFrance
  2. 2.Electrochemical Signaling in Development and DiseaseMax-Delbrück-Center for Molecular Medicine (MDC)Berlin-BuchGermany
  3. 3.Department of Animal Physiology, Faculty of Sciences and TechnicsUniversity of Abomey-CalaviCotonouBenin
  4. 4.Department of ChemistryDokuz Eylül UniversityIzmirTurkey
  5. 5.ESPACE-DEV, UMR UG UA UM IRDMontpellierFrance

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