Anthracycline anticancer agents, such as daunorubicin and doxorubicin, rank among the most effective and widely used anticancer drugs. However, their benefit is markedly reduced by the risk of severe cardiotoxicity. Anthracyclines undergo metabolic reduction of the side chain carbonyl group, producing hydroxy metabolites implicated in the cardiotoxicity. This study investigated toxicity, metabolism and cellular disposition of daunorubicin and its hydroxy metabolite, daunorubicinol, in isolated rat neonatal cardiomyocytes. Daunorubicin induced concentration-dependent cytotoxicity, whereas the toxicity of exogenously administered daunorubicinol was significantly lower despite induction of similar DNA damage. UHPLC-MS analyses revealed that daunorubicin rapidly penetrates cardiomyocytes and is metabolized to daunorubicinol, which is then released from the cells. The intracellular concentration of daunorubicinol was consistently lower than that of daunorubicin, indicating a reduced tendency for daunorubicinol to accumulate in cardiomyocytes. P-glycoprotein 1 has been shown to actively facilitate the efflux of both daunorubicin and daunorubicinol from cardiomyocytes. Dexrazoxane, the only approved agent for anthracycline cardiotoxicity prevention, did not affect the cellular metabolism or disposition of daunorubicin or its hydroxy metabolite, but it effectively reduced not only daunorubicin-induced cardiotoxicity, but also provided protection against the lower toxicity of daunorubicinol. Moreover, dexrazoxane reduced DNA damage induced by both daunorubicin and its hydroxy metabolite. These findings suggest that daunorubicin is the primary driver of cardiomyocyte cytotoxicity, while its hydroxy metabolite, daunorubicinol, plays a more limited role, challenging the notion that it serves as a significant toxic reservoir.

Department of Organic and Bioorganic Chemistry Faculty of Pharmacy in Hradec Králové Charles University Hradec Králové Czech Republic

Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Faculty of Pharmacy in Hradec Králové Charles University Hradec Králové Czech Republic

Faculty of Medicine in Hradec Králové Department of Pharmacology Charles University Hradec Králové Czech Republic

Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Charles University Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic

Zobrazit více v PubMed

Al-Otaibi TK, Weitzman B, Tahir UA, Asnani A (2022) Genetics of anthracycline-associated cardiotoxicity. Front Cardiovasc Med 9:867873. 10.3389/fcvm.2022.867873 PubMed PMC

Austin CA, Lee KC, Swan RL et al (2018) TOP2B: the first thirty years. Int J Mol Sci. 10.3390/ijms19092765 PubMed PMC

Ax W, Soldan M, Koch L, Maser E (2000) Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction. Biochem Pharmacol 59(3):293–300. 10.1016/s0006-2952(99)00322-6 PubMed

Bains OS, Szeitz A, Lubieniecka JM et al (2013) A correlation between cytotoxicity and reductase-mediated metabolism in cell lines treated with doxorubicin and daunorubicin. J Pharmacol Exp Ther 347(2):375–387. 10.1124/jpet.113.206805 PubMed

Bavlovic Piskackova H, Kollarova-Brazdova P, Kucera R, Machacek M, Pedersen-Bjergaard S, Sterbova-Kovarikova P (2021a) The electromembrane extraction of pharmaceutical compounds from animal tissues. Anal Chim Acta 1177:338742. 10.1016/j.aca.2021.338742 PubMed

Bavlovic Piskackova H, Oiestad EL, Vanova N, Lengvarska J, Sterbova-Kovarikova P, Pedersen-Bjergaard S (2021b) Electromembrane extraction of anthracyclines from plasma: comparison with conventional extraction techniques. Talanta 223(Pt 2):121748. 10.1016/j.talanta.2020.121748 PubMed

Berg SL, Reid J, Godwin K et al (1999) Pharmacokinetics and cerebrospinal fluid penetration of daunorubicin, idarubicin, and their metabolites in the nonhuman primate model. J Pediatr Hematol Oncol 21(1):26–30 PubMed

Bernardini N, Giannessi F, Bianchi F et al (1991) Comparative activity of doxorubicin and its major metabolite, doxorubicinol, on V79/AP4 fibroblasts: a morphofunctional study. Exp Mol Pathol 55(3):238–250. 10.1016/0014-4800(91)90004-h PubMed

Bhatia S (2020) Genetics of anthracycline cardiomyopathy in cancer survivors: JACC: cardiooncology state-of-the-art review. JACC CardioOncol 2(4):539–552. 10.1016/j.jaccao.2020.09.006 PubMed PMC

Booth LK, Redgrave RE, Folaranmi O, Gill JH, Richardson GD (2022) Anthracycline-induced cardiotoxicity and senescence. Front Aging 3:1058435. 10.3389/fragi.2022.1058435 PubMed PMC

Boucek RJ Jr., Olson RD, Brenner DE, Ogunbunmi EM, Inui M, Fleischer S (1987) The major metabolite of doxorubicin is a potent inhibitor of membrane-associated ion pumps. A correlative study of cardiac muscle with isolated membrane fractions. J Biol Chem 262(33):15851–15856 PubMed

Callies S, de Alwis DP, Harris A et al (2003) A population pharmacokinetic model for paclitaxel in the presence of a novel P-gp modulator, Zosuquidar trihydrochloride (LY335979). Br J Clin Pharmacol 56(1):46–56. 10.1046/j.1365-2125.2003.01826.x PubMed PMC

Callies S, de Alwis DP, Mehta A, Burgess M, Aarons L (2004) Population pharmacokinetic model for daunorubicin and daunorubicinol coadministered with zosuquidar.3HCl (LY335979). Cancer Chemother Pharmacol 54(1):39–48. 10.1007/s00280-004-0775-4 PubMed

Cvetkovic RS, Scott LJ (2005) Dexrazoxane : a review of its use for cardioprotection during anthracycline chemotherapy. Drugs 65(7):1005–1024. 10.2165/00003495-200565070-00008 PubMed

Del Tacca M, Danesi R, Ducci M, Bernardini C, Romanini A (1985) Might adriamycinol contribute to adriamycin-induced cardiotoxicity? Pharmacol Res Commun 17(11):1073–1084. 10.1016/0031-6989(85)90113-4 PubMed

Dempke WCM, Zielinski R, Winkler C, Silberman S, Reuther S, Priebe W (2023) Anthracycline-induced cardiotoxicity - are we about to clear this hurdle? Eur J Cancer 185:94–104. 10.1016/j.ejca.2023.02.019 PubMed

Evans CJ, Phillips RM, Jones PF et al (2009) A mathematical model of doxorubicin penetration through multicellular layers. J Theor Biol 257(4):598–608. 10.1016/j.jtbi.2008.11.031 PubMed

Forrest GL, Gonzalez B, Rivera H (1998) Daunorubicin-induced cardiotoxicity is decreased in transgenic mice with heart specific expression of human carbonyl reductases. vol 39. American Association for Cancer Research, Pharmacology and experimental therapeutics, p 597

Forrest GL, Gonzalez B, Tseng W, Li X, Mann J (2000) Human carbonyl reductase overexpression in the heart advances the development of doxorubicin-induced cardiotoxicity in transgenic mice. Cancer Res 60(18):5158–5164 PubMed

Gianni L, Herman EH, Lipshultz SE, Minotti G, Sarvazyan N, Sawyer DB (2008) Anthracycline cardiotoxicity: from bench to bedside. J Clin Oncol 26(22):3777–3784. 10.1200/JCO.2007.14.9401 PubMed PMC

Guo Y, Pu WT (2020) Cardiomyocyte maturation: new phase in development. Circ Res 126(8):1086–1106. 10.1161/CIRCRESAHA.119.315862 PubMed PMC

Huang KM, Hu S, Sparreboom A (2018) Drug transporters and anthracycline-induced cardiotoxicity. Pharmacogenomics 19(11):883–888. 10.2217/pgs-2018-0056 PubMed

Jirkovska A, Karabanovich G, Kubes J et al (2021) Structure-activity relationship study of dexrazoxane analogues reveals ICRF-193 as the most potent bisdioxopiperazine against anthracycline toxicity to cardiomyocytes due to its strong topoisomerase IIbeta interactions. J Med Chem 64(7):3997–4019. 10.1021/acs.jmedchem.0c02157 PubMed

Jirkovsky E, Jirkovska A, Bavlovic-Piskackova H et al (2021) Clinically translatable prevention of anthracycline cardiotoxicity by dexrazoxane is mediated by topoisomerase II beta and not metal chelation. Circ Heart Fail 14(11):e008209. 10.1161/CIRCHEARTFAILURE.120.008209 PubMed

Jong J, Pinney JR, Packard RRS (2022) Anthracycline-induced cardiotoxicity: from pathobiology to identification of molecular targets for nuclear imaging. Front Cardiovasc Med 9:919719. 10.3389/fcvm.2022.919719 PubMed PMC

Kaiserova H, Simunek T, van der Vijgh WJ, Bast A, Kvasnickova E (2007) Flavonoids as protectors against doxorubicin cardiotoxicity: role of iron chelation, antioxidant activity and inhibition of carbonyl reductase. Biochim Biophys Acta 1772(9):1065–1074. 10.1016/j.bbadis.2007.05.002 PubMed

Keizer HG, Pinedo HM, Schuurhuis GJ, Joenje H (1990) Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity. Pharmacol Ther 47(2):219–231. 10.1016/0163-7258(90)90088-j PubMed

Lazarowski AJ, Garcia Rivello HJ, Vera Janavel GL et al (2005) Cardiomyocytes of chronically ischemic pig hearts express the MDR-1 gene-encoded P-glycoprotein. J Histochem Cytochem 53(7):845–850. 10.1369/jhc.4A6542.2005 PubMed

Leger K, Slone T, Lemler M et al (2015) Subclinical cardiotoxicity in childhood cancer survivors exposed to very low dose anthracycline therapy. Pediatr Blood Cancer 62(1):123–127. 10.1002/pbc.25206 PubMed

Lenco J, Lencova-Popelova O, Link M et al (2015) Proteomic investigation of embryonic rat heart-derived H9c2 cell line sheds new light on the molecular phenotype of the popular cell model. Exp Cell Res 339(2):174–186. 10.1016/j.yexcr.2015.10.020 PubMed

Matyjaszczyk K, Kolonko M, Gonciarz-Dytman A et al (2017) Effects of structural modification of the daunosamine moiety of anthracycline antibiotics on pK(a) values determined by capillary zone electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci 1060:44–52. 10.1016/j.jchromb.2017.04.038 PubMed

McGowan JV, Chung R, Maulik A, Piotrowska I, Walker JM, Yellon DM (2017) Anthracycline chemotherapy and cardiotoxicity. Cardiovasc Drugs Ther 31(1):63–75. 10.1007/s10557-016-6711-0 PubMed PMC

Meissner K, Sperker B, Karsten C et al (2002) Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy. J Histochem Cytochem 50(10):1351–1356. 10.1177/002215540205001008 PubMed

Menna P, Salvatorelli E, Gianni L, Minotti G (2008) Anthracycline cardiotoxicity. Top Curr Chem 283:21–44. 10.1007/128_2007_11 PubMed

Menna P, Paz OG, Chello M, Covino E, Salvatorelli E, Minotti G (2012) Anthracycline cardiotoxicity. Expert Opin Drug Saf 11(Suppl 1):S21–S36. 10.1517/14740338.2011.589834 PubMed

Minotti G, Recalcati S, Mordente A et al (1998) The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium. FASEB J 12(7):541–552. 10.1096/fasebj.12.7.541 PubMed

Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56(2):185–229. 10.1124/pr.56.2.6 PubMed

Mordente A, Meucci E, Martorana GE, Giardina B, Minotti G (2001) Human heart cytosolic reductases and anthracycline cardiotoxicity. IUBMB Life 52(1–2):83–88. 10.1080/15216540252774829 PubMed

Mordente A, Minotti G, Martorana GE, Silvestrini A, Giardina B, Meucci E (2003) Anthracycline secondary alcohol metabolite formation in human or rabbit heart: biochemical aspects and pharmacologic implications. Biochem Pharmacol 66(6):989–998. 10.1016/s0006-2952(03)00442-8 PubMed

Mordente A, Silvestrini A, Martorana GE, Tavian D, Meucci E (2015) Inhibition of anthracycline alcohol metabolite formation in human heart cytosol: a potential role for several promising drugs. Drug Metab Dispos 43(11):1691–1701. 10.1124/dmd.115.065110 PubMed

Nguyen PH, Sigdel KP, Schaefer KG, Mensah GAK, King GM, Roberts AG (2020) The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences. Biochem Pharmacol 174:113813. 10.1016/j.bcp.2020.113813 PubMed PMC

Olson RD, Mushlin PS, Brenner DE et al (1988) Doxorubicin cardiotoxicity may be caused by its metabolite, doxorubicinol. Proc Natl Acad Sci U S A 85(10):3585–3589. 10.1073/pnas.85.10.3585 PubMed PMC

Ozols RF, Willson JK, Weltz MD, Grotzinger KR, Myers CE, Young RC (1980) Inhibition of human ovarian cancer colony formation by adriamycin and its major metabolites. Cancer Res 40(11):4109–4112 PubMed

Peters JH, Gordon GR, Kashiwase D, Acton EM (1981) Tissue distribution of doxorubicin and doxorubicinol in rats receiving multiple doses of doxorubicin. Cancer Chemother Pharmacol 7(1):65–69. 10.1007/BF00258216 PubMed

Peterson C, Trouet A (1978) Transport and storage of daunorubicin and doxorubicin in cultured fibroblasts. Cancer Res 38(12):4645–4649 PubMed

Petrelli F, Borgonovo K, Cabiddu M, Ghilardi M, Barni S (2012) Risk of anti-EGFR monoclonal antibody-related hypomagnesemia: systematic review and pooled analysis of randomized studies. Expert Opin Drug Saf 11(Suppl 1):S9–S19. 10.1517/14740338.2011.606213 PubMed

Platel D, Bonoron-Adele S, Robert J (2001) Role of daunorubicinol in daunorubicin-induced cardiotoxicity as evaluated with the model of isolated perfused rat heart. Pharmacol Toxicol 88(5):250–254. 10.1034/j.1600-0773.2001.d01-112.x PubMed

Qiao X, van der Zanden SY, Wander DPA et al (2020) Uncoupling DNA damage from chromatin damage to detoxify doxorubicin. Proc Natl Acad Sci U S A 117(26):15182–15192. 10.1073/pnas.1922072117 PubMed PMC

Qiao X, van der Zanden SY, Li X et al (2024) Diversifying the anthracycline class of anti-cancer drugs identifies aclarubicin for superior survival of acute myeloid leukemia patients. Mol Cancer 23(1):120. 10.1186/s12943-024-02034-7 PubMed PMC

Regev R, Eytan GD (1997) Flip-flop of doxorubicin across erythrocyte and lipid membranes. Biochem Pharmacol 54(10):1151–1158. 10.1016/s0006-2952(97)00326-2 PubMed

Regev R, Yeheskely-Hayon D, Katzir H, Eytan GD (2005) Transport of anthracyclines and mitoxantrone across membranes by a flip-flop mechanism. Biochem Pharmacol 70(1):161–169. 10.1016/j.bcp.2005.03.032 PubMed

Reis-Mendes AF, Sousa E, de Lourdes BM, Costa VM (2015) The role of the metabolism of anticancer drugs in their induced-cardiotoxicity. Curr Drug Metab 17(1):75–90. 10.2174/1389200216666151103114926 PubMed

Reis-Mendes A, Vitorino-Oliveira C, Ferreira M et al (2024) Comparative in vitro study of the cytotoxic effects of doxorubicin’s main metabolites on cardiac AC16 cells versus the parent drug. Cardiovasc Toxicol 24(3):266–279. 10.1007/s12012-024-09829-6 PubMed PMC

Robert J, Gianni L (1993) Pharmacokinetics and metabolism of anthracyclines. Cancer Surv 17:219–252 PubMed

Saleh Y, Abdelkarim O, Herzallah K, Abela GS (2021) Anthracycline-induced cardiotoxicity: mechanisms of action, incidence, risk factors, prevention, and treatment. Heart Fail Rev 26(5):1159–1173. 10.1007/s10741-020-09968-2 PubMed

Sasaya M, Wada I, Shida M et al (1998) Uptake of doxorubicin by cultured kidney epithelial cells LLC-PK1. Biol Pharm Bull 21(5):527–529. 10.1248/bpb.21.527 PubMed

Sawicki KT, Sala V, Prever L, Hirsch E, Ardehali H, Ghigo A (2021) Preventing and treating anthracycline cardiotoxicity: new insights. Annu Rev Pharmacol Toxicol 61:309–332. 10.1146/annurev-pharmtox-030620-104842 PubMed

Schroder JK, Kasimir-Bauer S, Seeber S, Scheulen ME (2000) In vitro effect of multidrug resistance modifiers on idarubicinol efflux in blasts of acute myeloid leukemia. J Cancer Res Clin Oncol 126(2):111–116. 10.1007/s004320050019 PubMed PMC

Simunek T, Sterba M, Popelova O et al (2008) Pyridoxal isonicotinoyl hydrazone (PIH) and its analogs as protectants against anthracycline-induced cardiotoxicity. Hemoglobin 32(1–2):207–215. 10.1080/03630260701680276 PubMed

Sorf A, Novotna E, Hofman J et al (2019) Cyclin-dependent kinase inhibitors AZD5438 and R547 show potential for enhancing efficacy of daunorubicin-based anticancer therapy: interaction with carbonyl-reducing enzymes and ABC transporters. Biochem Pharmacol 163:290–298. 10.1016/j.bcp.2019.02.035 PubMed

Sterba M, Popelova O, Vavrova A et al (2013) Oxidative stress, redox signaling, and metal chelation in anthracycline cardiotoxicity and pharmacological cardioprotection. Antioxid Redox Signal 18(8):899–929. 10.1089/ars.2012.4795 PubMed PMC

Suter TM, Ewer MS (2013) Cancer drugs and the heart: importance and management. Eur Heart J 34(15):1102–1111. 10.1093/eurheartj/ehs181 PubMed

Uddin ME, Moseley A, Hu S, Sparreboom A (2022) Contribution of membrane transporters to chemotherapy-induced cardiotoxicity. Basic Clin Pharmacol Toxicol 130(Suppl 1):36–47. 10.1111/bcpt.13635 PubMed

van Asperen J, van Tellingen O, Tijssen F, Schinkel AH, Beijnen JH (1999) Increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr1a P-glycoprotein. Br J Cancer 79(1):108–113. 10.1038/sj.bjc.6690019 PubMed PMC

Vavrova A, Simunek T (2012) DNA topoisomerase IIbeta: a player in regulation of gene expression and cell differentiation. Int J Biochem Cell Biol 44(6):834–837. 10.1016/j.biocel.2012.03.005 PubMed

Vavrova A, Jansova H, Mackova E et al (2013) Catalytic inhibitors of topoisomerase II differently modulate the toxicity of anthracyclines in cardiac and cancer cells. PLoS ONE 8(10):e76676. 10.1371/journal.pone.0076676 PubMed PMC

Veitch ZW, Guo B, Hembruff SL et al (2009) Induction of 1C aldoketoreductases and other drug dose-dependent genes upon acquisition of anthracycline resistance. Pharmacogenet Genomics 19(6):477–488. 10.1097/FPC.0b013e32832c484b PubMed

Vejpongsa P, Yeh ET (2014) Prevention of anthracycline-induced cardiotoxicity: challenges and opportunities. J Am Coll Cardiol 64(9):938–945. 10.1016/j.jacc.2014.06.1167 PubMed

WHO electronic Essential Medicines List (eEML) (2023) https://list.essentialmeds.org/. Accessed 6 Dec 2024

Zeng X, Cai H, Yang J, Qiu H, Cheng Y, Liu M (2019) Pharmacokinetics and cardiotoxicity of doxorubicin and its secondary alcohol metabolite in rats. Biomed Pharmacother 116:108964. 10.1016/j.biopha.2019.108964 PubMed

Zhang S, Liu X, Bawa-Khalfe T et al (2012) Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med 18(11):1639–1642. 10.1038/nm.2919 PubMed