Early and delayed cardioprotective intervention with dexrazoxane each show different potential for prevention of chronic anthracycline cardiotoxicity in rabbits

Toxicology 311 (2013) 191–204

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Early and delayed cardioprotective intervention with dexrazoxane each show different potential for prevention of chronic anthracycline cardiotoxicity in rabbits Eduard Jirkovsky´ a , Olga Lenˇcová-Popelová a , Miloˇs Hroch a,b , Michaela Adamcová c , f ˇ unek ˚ Yvona Mazurová d , Jaroslava Vávrová e , Stanislav Miˇcuda a , Tomáˇs Sim , a a,∗ ˇ Vladimír Gerˇsl , Martin Stˇerba Department of Pharmacology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Sˇ imkova 870, Hradec Králové 500 38, Czech Republic Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University in Prague, Sˇ imkova 870, Hradec Králové 500 38, Czech Republic c Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Sˇ imkova 870, Hradec Králové 500 38, Czech Republic d Department of Histology and Embryology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Sˇ imkova 870, Hradec Králové 500 38, Czech Republic e Department of Clinical Biochemistry and Diagnosis, University Hospital Hradec Králové, Sokolská 581, Hradec Králové 500 05, Czech Republic f Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic a

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Article history: Received 10 May 2013 Received in revised form 25 June 2013 Accepted 26 June 2013 Available online 4 July 2013 Keywords: Dexrazoxane Cardioprotection Anthracycline Cardiotoxicity Heart failure Mechanisms

a b s t r a c t Despite incomplete understanding to its mechanism of action, dexrazoxane (DEX) is still the only clearly effective cardioprotectant against chronic anthracycline (ANT) cardiotoxicity. However, its clinical use is currently restricted to patients exceeding significant ANT cumulative dose (300 mg/m2 ), although each ANT cycle may induce certain potentially irreversible myocardial damage. Therefore, the aim of this study was to compare early and delayed DEX intervention against chronic ANT cardiotoxicity and study the molecular events involved. The cardiotoxicity was induced in rabbits with daunorubicin (DAU; 3 mg/kg/week for 10 weeks); DEX (60 mg/kg) was administered either before the 1st or 7th DAU dose (i.e. after ≈300 mg/m2 cumulative dose). While both DEX administration schedules prevented DAU-induced premature deaths and severe congestive heart failure, only the early intervention completely prevented the left ventricular dysfunction, myocardial morphological changes and mitochondrial damage. Further molecular analyses did not support the assumption that DEX cardioprotection is based and directly proportional to protection from DAU-induced oxidative damage and/or deletions in mtDNA. Nevertheless, DAU induced significant up-regulation of heme oxygenase 1 pathway while heme synthesis was inversely regulated and both changes were schedule-of-administration preventable by DEX. Early and delayed DEX interventions also differed in ability to prevent DAU-induced down-regulation of expression of mitochondrial proteins encoded by both nuclear and mitochondrial genome. Hence, the present functional, morphological as well as the molecular data highlights the enormous cardioprotective effects of DEX and provides novel insights into the molecular events involved. Furthermore, the data suggests that currently recommended delayed intervention may not be able to take advantage of the full cardioprotective potential of the drug. © 2013 Published by Elsevier Ireland Ltd.

Abbreviations: ALAS1, 5-aminolevulinate synthase 1; ANT, anthracycline; BVR A, biliverdin reductase A; COX1, COX4, mitochondrial and nuclear genome-encoded complex IV subunits, respectively; DAU, daunorubicin; DEX, dexrazoxane; FU, post-treatment follow up; GSH, reduced glutathione; GSSG, oxidized glutathione; HIF1␣, hypoxia-inducible factor 1␣; HO1, heme oxygenase 1; LV FS, left ventricular fractional shortening; MDA, malondialdehyde; MnSOD, mitochondrial superoxide dismutase; mtDNA, mitochondrial DNA; ND1, ND4 mitochondrial genome-encoded complex I subunits; nDNA, nuclear DNA; NDUFS2, nuclear genome-encoded complex I subunit; NOX2, NOX4, NADPH oxidases 2 and 4; NQO1, NAD(P)H dehydrogenase [quinone] 1; NRF1, nuclear respiratory factor 1; Nrf2, nuclear factor erythroid 2-related factor 2; PRDX3, peroxiredoxin 3; TFAM, mitochondrial transcription factor A. ∗ Corresponding author. Tel.: +420 495 816 312; fax: +420 495 513 597. ˇ erba). E-mail address: [email protected] (M. Stˇ 0300-483X/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.tox.2013.06.012

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1. Introduction Anthracycline (ANT) antibiotics (e.g. doxorubicin, daunorubicin or epirubicin) continue to form a backbone of anticancer therapy in many hematological and solid malignancies. However, all ANTs are also known for their risk of chronic cardiotoxicity which may result in cardiomyopathy and heart failure with either early or late onset. Current clinical guidelines attempt to minimize the risks by limitation of the total cumulative dose of ANTs and by implementation of non-invasive monitoring of cardiac function (Aapro et al., 2011; Menna et al., 2012). Nevertheless, it is becoming clear that there is no completely safe cumulative dose of ANTs. Instead, it is now appreciated that each ANT cycle likely induces certain molecular and ultrastructural damage to the myocardium which is largely irreversible and manifests itself after accumulation of the damage from multiple cycles (Ewer and Benjamin, 2006). Hence, patients undergoing ANT treatment may carry clinical or more often subclinical cardiac burden to their post-cancer life (Aapro et al., 2011; Ewer and Benjamin, 2006; Menna et al., 2012). The optimal approach to management of ANT cardiotoxicity risk is its effective prevention. Hence, the search for an effective pharmacological cardioprotectant began soon after the early clinical trials recognizing this complication. More than 40 years of intensive research yielded many disappointing outcomes which also concerned classic antioxidants including vitamin E or acetylcysteine (Dresdale et al., 1982; Legha et al., 1982; Myers et al., 1983; Sterba et al., 2013). However, major success has also been achieved in this field-development of dexrazoxane (DEX) which is still the only drug which has been convincingly demonstrated to provide effective cardioprotection in both clinical and experimental settings (Cvetkovic and Scott, 2005; van Dalen et al., 2011). Furthermore, DEX has been shown to induce effective protection from both ANT-induced degenerative changes and apoptotic death of cardiomyocytes (Popelova et al., 2009; Sawyer et al., 1999). Traditionally, DEX is believed to be a pro-drug of iron chelating metabolite (ADR-925) which prevents participation of iron in ANT redox cycling in the heart (Cvetkovic and Scott, 2005; Hasinoff and Herman, 2007). More recently, DEX has been proposed to specifically protect mitochondrial DNA (mtDNA) from oxidative stress-induced common deletions and impaired expression of mtDNA-encoded respiratory chain subunits (Lebrecht et al., 2007). However, stronger and more selective intracellular iron chelators failed to provide better or at least comparable cardioprotection as DEX in chronic ANT cardiotoxicity models (Sterba et al., 2013) which argues against this hypothesis and some investigators have questioned even the whole pro-drug concept (Hasinoff and Herman, 2007; Lyu et al., 2007; Zhang et al., 2012). Thus, the mechanisms responsible for effective cardioprotection provided by DEX are poorly understood. Despite its well-evidenced cardioprotective effects, DEX is currently recommended to be only used when a critical cumulative ANT dose of 300 mg/m2 is achieved (Hensley et al., 2009). This recommendation was originally driven by outcomes of a single clinical trial suggesting significant impact of DEX on objective response rate of breast cancer to the ANT-based chemotherapy (Swain et al., 1997b). Although the interpretation of this finding is far from being straightforward (Swain and Vici, 2004) and an independent metaanalysis of all randomized clinical trials has failed to find any evidence for this hypothesis (van Dalen et al., 2011), the guidelines for clinical use of DEX remain still the same. Indeed, even the recommended “delayed” administration of DEX showed a benefit in a clinical trial (Swain et al., 1997a), however, there is a considerable lack of understanding what happens to the myocardium in this settings as compared to the “early” intervention, where DEX is administered before each ANT cycle. Furthermore, such

investigation could shed more light on molecular events important for effective cardioprotection. Therefore, the aim of the present study was to evaluate cardioprotective effects of DEX administered either before each daunorubicin (DAU) dose or with significant delay (from cumulative DAU dose of 300 mg/m2 ) on a well-established rabbit model of chronic ANT cardiotoxicity. Furthermore, functional, morphological as well as molecular aspects of both cardioprotective interventions were investigated. 2. Materials and methods All chemicals were purchased from Sigma-Aldrich (MO, USA) unless stated otherwise. 2.1. Study design and animals treatment The study used well-established model of chronic ANT cardiotoxicity (Gersl et al., 1999; Popelova et al., 2009; Simunek et al., 2004). All animal handling and procedures were approved and supervised by Ethical Committee of the Faculty of Medicine in Hradec Králové. Chinchilla male rabbits (n = 77, ∼3.5 kg, ∼0.2 m2 body surface area) were randomized to treatments as follows: cardiotoxicity was induced with daunorubicin (3 mg/kg/week, i.v., for 10 weeks; the DAU group, n = 27). DEX (60 mg/kg, i.p. 30 min before DAU) was administered either from the 1st DAU dose (early DEX intervention, DD1 group, n = 16) or from the 7th one (delayed DEX intervention, DD7 group, n = 15), i.e. after exceeding cumulative DAU dose of 300 mg/m2 as calculated according to Ward (2008). Controls received saline (1 mL/kg/week, i.v., for 10 weeks, CTR group n = 19). A week after the last administration, animals in each group were randomized for sacrifice or for a 10-week post-treatment follow up (FU; DAU, n = 11; CTR, n = 10; DD1 , n = 8; DD7 , n = 8). Mortality was determined during the treatment period, whereas during the FU period animals were sacrificed whenever weekly echocardiography examination showed left ventricular fractional shortening (LV FS) to be lower than 20%, which indicates decompensated cardiac failure, to avoid loss of myocardial samples because of sudden deaths. All non-invasive procedures and blood sampling were performed under light anesthesia mixture of ketamine (30 mg/kg) and midazolam (2.5 mg/kg), while pentobarbital (30 mg/kg) was used for invasive hemodynamic measurements and animal sacrifice. After animal overdose, a gross autopsy was performed and the heart was rapidly excised and retrogradely perfused with ice-cold saline. Transversal sections of whole heart were cut for histological analysis, while the rest of the left ventricle (LV) was frozen in liquid nitrogen, pulverized under liquid nitrogen and stored at −80 ◦ C. 2.2. Cardiac function examinations Echocardiography was used to examine LV function during the study (Vivid 4 equipped with 10 MHz probe, GE Healthcare, UK). Left parasternal approach was employed to perform guided M-mode examinations in long and short axis view. LV end-systolic (LVESD) and LV end-diastolic (LVEDD) diameters were determined and LV fractional shortening (LV FS) was calculated as follows: LV FS (%) = ((LVEDDLVESD)/LVEDD) × 100. At the end of the study, invasive LV hemodynamic measurements were performed via A. carotis sinistra using Micro-Tip pressure catheter (2.3F, Millar Instruments, TX, USA) connected to a data acquisition system (Powerlab, ADInstruments Pty., Australia). The first derivative of the LV pressure change in the isovolumic phase of the systole and diastole (index dP/dtmax and dP/dtmin , respectively) were calculated using Chart 5.4.2 software (ADInstruments Pty.). All measurements were performed after the equilibration interval (15 min) to stabilize the animals after the preparation. 2.3. Plasma troponin T determination Blood samples were obtained before the 1st, 5th, 7th, 10th drug administration, at the end of the study and in the 13th, 15th, 17th and 21st week in the FU period. Concentrations of cardiac troponin T in plasma were determined using Elecsys Troponin T hs assay kit (Roche Diagnostics, Switzerland) with a limit of detection of 0.003 ␮g/L. 2.4. Histopathological examinations During autopsy, the blocks of heart tissue (each approx. 3 mm thick) were transversely cut off at the level under the atrioventricular septum, post-fixed by immersion in 4% neutral formaldehyde for 3 days and embedded in paraffin. Serial sections (6 ␮m thick), were stained with hematoxylin and eosin (H&E) and Masson’s blue trichrome. Photomicrographs were made using the microscope Olympus BX 51 equipped with the digital camera DP 70 (Olympus, Japan) and Quick Photo Camera 2.3 software (Promicra, Czech Republic).

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Fig. 1. The effects of dexrazoxane interventions on daunorubicin-induced general toxicity, cardiac dysfunction and biomarkers of cardiac damage. (A) Body weight gain in comparison to the initial values. (B) Echocardiography-assessed left ventricular systolic function (FS—fractional shortening). (C) Catheterizationassessed index of the left ventricular systolic (dP/dtmax ) and (D) diastolic function (dP/dtmin ). (E) Plasma concentrations of cardiac troponin T as a marker of cardiac damage. The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively or as bars with median and interquartile range. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR), “D” (daunorubicin; DAU) and “1” (early dexrazoxane intervention; DD1 ). Significant change (paired t-tests, p < 0.05) between the treatment and post-treatment period is indicated by “#“, while “*” indicates significant change as compared with the initial values.

2.5. Measurements of oxidative stress markers Measurements were performed in LV samples homogenized as described previously (Jirkovsky et al., 2012; Popelova et al., 2009). Glutathione content was measured according to Kand’ar et al. (2007). This is as a selective HPLC method based on derivatization of reduced glutathione (GSH) with o-phthalaldehyde yielding a fluorescent derivative. For selective measurement of oxidized glutathione (GSSG), N-ethylmaleimide pretreatment was employed before the o-phthalaldehyde

derivatization to remove GSH from the samples. For measurement of MDA content a selective TBARS-independent HPLC method based on the derivatization of MDA with 2,4-dinitrophenylhydrazine was used (Pilz et al., 2000). 2.6. Determination of total citrate synthase activity The citrate synthase activity was assayed according to De Sousa et al. (1999) as the enzyme-coupled formation of citric acid from acetyl-CoA and oxaloacetic

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Fig. 2. Histological examination of the left ventricular myocardium. (Aa) In the control group, normal structure of the myocardium was observed both at the end of treatment and (Ab) after the 10 week post-treatment follow up. (Ba) In the DAU group, foci of cardiomyocytes showing profound degeneration were found at the end of treatment. Significant morphological changes comprised mainly from myofibrillar disarray to profound loss of myofibrils which together with cytoplasmic vacuolization (asterisk) resulted in cell death of many cardiomyocytes. (Bb) The similar findings were found in the post-treatment follow up of the DAU group; however, the degenerative changes tended to be slightly less expressed, while replacement fibrosis (cross) up to the formation of fine scars (double-cross) seemed more frequent. (C) Almost normal structure of the myocardium was typically found at the end of treatment in animals undergoing early cardioprotective intervention with dexrazoxane (DD1 group). Degenerative changes in cardiomyocytes were only rarely observed and manifested mostly by the initial stage of myofibrillar break-down (arrow) in few isolated cardiomyocytes. (D) Very similar findings were also observed after the post-treatment follow up of animals with early DEX intervention. Cardiomyocytes with signs of degenerative changes were very rare again, and if found, they usually showed mild to moderate loss of myofibrils and cytosolic vacuolization (triangle). (E) Delayed cardioprotective intervention with DEX (DD7 group) showed markedly different results already by the end of treatment. Here, degenerative changes in cardiomyocytes were clearly recognizable (asterisk), although they were less extensive than in the DAU-alone group. Significant replacement fibrosis (cross, double-cross) was also present in affected foci of cardiomyocytes suggesting that reparation process took place during the DEX co-treatment. (F) Post-treatment follow up of animals with delayed DEX intervention was associated with continuous progression of the healing process in the myocardium (the formation of fine fibrotic scars—double-cross). Degenerating cardiomyocytes were less frequent (asterisk), while the replacement fibrosis was spread throughout affected areas. Masson’s blue trichrome. Bar ((A)–(F)): 50 ␮m.

acid. Myocardial samples were homogenized in ice cold buffer (50 mg/mL, pH 8.7) containing 5 mmol/L HEPES, 1 mmol/L EGTA, 1 mmol/L DTT and 0.1% Triton X-100. Samples were homogenized with glass balls using Thermomixer Comfort (Eppendorf, Germany, 60 min, 1400 rpm at 4 ◦ C). Final homogenates were centrifuged for 5 min at 800 × g, 4 ◦ C and supernatants were stored at −80 ◦ C. 2.7. Nucleic acid isolation and reverse transcriptase PCR analysis Total RNA from the LV myocardium was isolated using TRI Reagent (SigmaAldrich, MO, USA) according to manufacturer’s protocol. The isolated RNA was converted into cDNA via a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). Gene expression was examined by quantitative real-time RT-PCR as described previously (Jirkovsky et al., 2012) with a 7500HT Fast RealTime PCR System (Applied Biosystems) using TaqMan Fast Universal PCR Master

Mix (Applied Biosystems) and commercially available qPCR assays (see Supplementary Table 1) obtained from Generi Biotech (Czech Republic) or Applied Biosystems. Expressions of target genes were normalized on the expression of HPRT1 (reference gene) using Pffafl method (Pfaffl, 2001). For analysis of mtDNA/nDNA change, total DNA was extracted from the LV myocardium using a DNeasy Blood&Tissue Kit (Qiagen, CA, USA) according to manufacturer’s protocol. Three mtDNA-encoded genes (ND1, ND4 and COX1) were used to analyze relative abundance of mtDNA using qPCR as describe previously (Jirkovsky et al., 2012). Averaged results were normalized on the relative abundance of nDNA-encoded leptin using the Pffafl method. 2.8. Analysis of mtDNA common deletions The presence of common deletions and related large rearrangements in mtDNA was analyzed using long-range PCR with custom designed and optimized primers

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Lithuania) was used to estimate the size of the PCR products and ethidium bromide was used for DNA detection. 2.9. Measurement of Nrf2 active form in the nuclear fraction The amount of transcriptionally active form of Nrf2 was determined in the LV nuclear extracts using a DNA-binding ELISA kit (TransAM Nrf2, Active Motif, CA, USA) according to the manufacturer’s recommendations. Nuclear extracts were isolated from LV myocardium using Dignam et al. (1983) protocol. Briefly, approximately 50 mg of the tissue was homogenized in the ice cold buffer A (10 mmol/L HEPES, 1.5 mmol/L MgCl2 , 10 mmol/L KCl, 0.5 mmol/L DTT, 0.005% NP40 (v/v), protease inhibitor cocktail, pH 7.9). After centrifugation, the pellet was collected and dissolved in a mixture of ice cold buffer B (5 mmol/L HEPES, 1.5 mmol/L MgCl2 , 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 26% (v/v) glycerol, pH 7.9) and 4.6 M NaCl) and treated with repeated sonication (1 min, 0.5 cycle, 50% amplitude and 1 min, 0.5 cycle, 100% amplitude; Ultrasonic Processor UP100H, Hielscher, Germany), incubated for 30 min on ice and centrifuged for 20 min at 16,000 × g. 2.10. Western Blot analysis Proteins in the nuclear fraction (preparation as described above in Section 2.9) were separated on TGX AnykD Precast Gels (Bio-Rad, CA, USA), and electrically transferred onto PVDF (Pall Corporation, NY, USA) using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). Mouse monoclonal anti-HIF1␣ (Abcam, UK; dilution 1:150) was used as primary antibody, and horseradish peroxidaseconjugated goat anti-mouse IgG (DakoCytomation, Denmark, dilution 1:1000) was used the secondary antibody. BM Chemiluminescence Blotting Substrate (Roche, Germany) was used for detection. Protein loading was controlled with Ponceau S staining of PVDF membranes. Densitometric quantification was performed using Quantity One software (Bio-Rad). 2.11. Determination of protein concentration Protein concentration was determined using a BCA Protein Assay Kit (SigmaAldrich, MO, USA) according manufacturer’s protocol. 2.12. Statistical analyses The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles unless stated otherwise to properly describe the data distribution independently on the data character. Statistical significance difference was set at p < 0.05 and determined using One Way ANOVA/ANOVA on Ranks or paired t-test/Wilcoxon Signed Rank Test according to the data character using software Sigmastat 3.5 (SPSS, USA).

3. Results 3.1. General toxicity

Fig. 3. Left ventricular oxidative stress markers. (A) Left ventricular oxidized-to-reduced glutathione ratio (GSSG/GSH). (B) HPLC determined oxidized glutathione content (GSSG). (C) HPLC determined total malondialdehyde content (a marker of lipoperoxidation) using TBARS-independent method. The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR), “D” (daunorubicin; DAU) and “1” (early dexrazoxane intervention; DD1 ). “#“indicates statistically significant change (One Way ANOVA/ANOVA on Ranks, p < 0.05) between the study periods within the groups receiving same treatments.

(5 -GGACTCTACTCGGGGATGA-3 and 3 -GGGTGTAGTTGTCTGGGTCTC-5 ) (Generi Biotech, Czech Republic) and QIAGEN LongRange PCR kit (Qiagen, CA, USA) according to the manufacturer’s recommendations. The assay amplifies mtDNA between 5487 and 14,946 bp yielding the 9460 bp PCR product. Time-temperature profile of the assay was as follow: 93 ◦ C for 3 min; 35 cycles: 93 ◦ C for 15 s, 61.5 ◦ C for 30 s, 68 ◦ C for 10.5 min; 68 ◦ C for 12 min. The PCR product was analyzed using 1% agarose gel electrophoresis (90 V for 40–60 min). GeneRuler 1 kb DNA Ladder (Fermentas,

Dexrazoxane co-treatment in both dosing schedules effectively prevented the onset of DAU-induced premature deaths in the treatment period (0 vs. 31%) and all DEX-treated animals survived till the end of the FU period without any apparent signs of poor health. This strikingly contrasted with the DAU group where the majority of the animals (82%) had to be sacrificed during the FU period according to the study protocol (FS < 20%) to prevent loss of myocardial samples due to the sudden death. DAU treatment precluded a significant weight gain in the treatment period (Fig. 1A) and similar results were found after the early DEX intervention (DD1 group), whereas animals undergoing the delayed one (DD7 group) performed slightly better. On the other hand, in the FU period, the animals receiving the early DEX intervention showed significantly higher body weight as compared to the DAU group, which was not the case of the delayed DEX intervention. In line with this, hydrothorax and ascites were frequently found during autopsy in the DAU group, especially in the FU period (in volumes up to 75 mL), whereas no significant fluid volume (>1 mL) was observed in any either of DEX-treated animals. 3.2. LV cardiac function Although both schedules of DEX treatment showed certain degree of cardioprotection, markedly better outcomes were

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Fig. 4. Gene expression and abundance of pro- and anti-oxidant proteins in the left ventricle myocardium. (A) Relative gene expression of NOX2 and (B) NOX4 (NADPH oxidases). (C) Relative abundance of transcriptionally active form of Nrf2 (nuclear factor erythroid 2-related factor 2) in the myocardial nuclear fraction. (D) Relative gene expression of MnSOD (mitochondrial superoxide dismutase), (E) PRDX3 (peroxiredoxin 3) and (F) NQO1 (NAD(P)H dehydrogenase [quinone] 1). The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR), “D” (daunorubicin; DAU) and “1” (early dexrazoxane intervention; DD1 ). “#“indicates statistically significant change (One Way ANOVA/ANOVA on Ranks, p < 0.05) between the study periods within the groups receiving same treatments.

obtained in the DD1 group receiving cardioprotectant with each DAU dose. In this schedule, DEX treatment completely prevented severe deterioration of LV systolic function as determined by both echocardiography (Fig. 1B) and catheterization (Fig. 1C). Of particular note, the same protection of systolic dysfunction was found in

the FU period which confirmed strong and long-lasting cardioprotection. Delayed DEX intervention in the DD7 group was associated with significant decline in the LV systolic function as determined by both approaches (Fig. 1B and C). This change persisted throughout the FU period, although it was less pronounced than in the

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Fig. 5. Expression of heme-metabolism related proteins in the left ventricular myocardium. Relative gene expression of (A) HO1 (heme oxygenase 1), (B) BVR A (biliverdin reductase A), (C) ferritin L subunit, (D) ALAS1 (5-aminolevulinate synthase 1) and (E) myoglobin in the left ventricular myocardium. The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR), “D” (daunorubicin; DAU) and “1” (early dexrazoxane intervention; DD1 ).

DAU group. Both DEX treatment regimens showed similar outcomes regarding prevention of DAU-induced diastolic dysfunction (Fig. 1D). 3.3. Cardiac troponin T as a marker of the cardiotoxicity Both DEX interventions significantly reduced plasma concentrations of cardiac troponin T not only in the treatment

period, but also in the FU period (Fig. 1E). Again, early DEX administration showed significantly better outcomes with no significant elevation above control values both in the treatment and post-treatment period. Although cardiac troponin T concentrations were nearly identical in the DAU and DD7 group in the 7th week (i.e. before the start of the delayed cardioprotective intervention), further rise of the biomarker was prevented in the latter group, although the values were still

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Fig. 6. Treatment-induced changes in hypoxia-inducible factor 1␣ in the left ventricular myocardium. Relative gene expression of (A) HIF1␣ (hypoxia-inducible factor 1␣) in the left ventricular myocardium and (B) relative protein abundance of HIF1␣ in the myocardial nuclear fraction. The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR), “D” (daunorubicin; DAU).

significantly higher than after the early DEX intervention (DD1 group). 3.4. DEX-afforded protection of the LV myocardium Histological examination of the LV myocardium in the DAU group documented characteristic degenerative changes at the end of treatment (esp. loss of myofibrils and cytoplasmic vacuolization, Fig. 2Ba) along with only limited healing process in a sense of replacement fibrosis. The similar pathological changes due to the DAU treatment were found also in the FU period (Fig. 2Bb), but intensity of degenerative changes tended to be somewhat less apparent, while replacement fibrosis became prominent, especially in animals surviving whole FU period. DEX co-treatment was able to either prevent or significantly reduce all above mentioned pathological changes. This was particularly true for the early intervention (Fig. 2C and D), where degenerative changes were absent or very scarce (concerning only a few isolated cardiomyocytes with mild to moderate alterations) and the myocardium was otherwise of

Fig. 7. Treatment-induced changes in parameters reflecting mitochondrial status in the left ventricular myocardium. (A) Citrate synthase activity. (B) Relative change in mitochondrial DNA/nuclear DNA ratio. Relative gene expression of (C) TFAM (transcription factor A, mitochondrial) in the left ventricular myocardium. The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR) and “D” (daunorubicin; DAU). “#” indicates statistically significant change (One Way ANOVA/ANOVA on Ranks, p < 0.05) between the study periods within the groups receiving same treatments.

normal morphology. Indeed, no distinct progression was recognizable in the FU period in this group. In contrast, the degenerative changes were evidently much more extensive and intensive in animals receiving the delayed DEX intervention (Fig. 2E and F). Apparent replacement fibrosis was developed already at the end of the treatment (Fig. 2E) and the character of this pathology headed

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Fig. 8. Expression of mitochondrial proteins in the left ventricular myocardium. Relative gene expression of (A) ND4 (mitochondrial DNA-encoded subunit of complex I), (B) COX1 (mitochondrial DNA-encoded subunit of complex IV), (C) NDUFS2 (nuclear DNA-encoded subunit of complex I), (D) COX4 (nuclear DNA-encoded subunit of complex IV), (E) ANT1 (adenine nucleotide translocase type 1), (F) NRF1 (nuclear respiratory factor 1) in left ventricular myocardium. The data are shown as medians with boxes and whiskers representing interquartile range and 5–95 percentiles, respectively. Statistical differences (One Way ANOVA/ANOVA on Ranks, p < 0.05) within each treatment period are displayed as follows: “C” (control; CTR) and “D” (daunorubicin; DAU). “#” indicates statistically significant change (One Way ANOVA/ANOVA on Ranks, p < 0.05) between the study periods within the groups receiving same treatments.

towards the chronic stage in the FU period (Fig. 2F). In comparison with the DAU group, delayed DEX intervention apparently prevented continuation of cardiomyocytes’ degeneration, which was reflected by lower number of severely damaged myocytes found

in this group. In addition, DEX intervention evidently supported the healing process which took place already during the treatment period and progressed further during the follow up, where its hallmarks became particularly prominent (Fig. 2F).

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3.5. LV oxidative stress and antioxidant defense Examination of the glutathione system in the LV myocardium showed no changes in the key parameter (GSSG/GSH ratio) in either of the studied groups (Fig. 3A). Nevertheless, presence of certain oxidative challenge could be found in the DAU group since GSSG increased significantly in the FU period (Fig. 3B), whereas no such change was present in DEX co-treated animals. Despite the excellent degree of cardioprotection found at the end of the treatment after the early DEX intervention, there was observed only minor and insignificant changes in the myocardial lipoperoxidation as compared to the DAU group (Fig. 3C). Furthermore, despite certain cardioprotective effects, the delayed DEX intervention had no impact on this parameter at this point in time. Worthy of note is the effect of DEX on myocardial lipoperoxidation which was more significant in the FU period, where markedly lower values of the MDA were found after the early DEX intervention (DD1 group) as compared to the DAU and DD7 group (Fig. 3C). Since NADPH oxidases (NOXs) were implicated in the oxidative stress and development of anthracycline cardiotoxicity, the expression of two main heart isoforms of NADPH oxidases (NOX2 and NOX4) was analyzed in this study. Interestingly, a significant induction of both isoforms was found after DAU treatment and these alterations persisted in the FU period (Fig. 4A and B). The early DEX administration completely prevented all these changes, whereas the delayed one was unable to do the same. Although signs of DAU-induced oxidative stress were observed in the study, we found no coordinated activation of master regulator of antioxidant response nuclear factor erythroid 2-related factor 2 (Nrf2) in the LV myocardium (Fig. 4C). The only increase in the Nrf2 active form was found after the delayed DEX intervention (DD7 group) at the end of the treatment, however, without significant response of its target genes (data not shown). On the contrary, some of the important Nrf2-target gene antioxidants as MnSOD, peroxiredoxin 3 and NAD(P)H dehydrogenase [quinone] 1 (NQO1) (Figs. 4D–F) were down-regulated due to DAU treatment and DEX administration effectively prevented these changes particularly in the early intervention settings (DD1 group). Although there was no coordinated up-regulation of the Nrf2 pathway, we found marked and persistent up-regulation of heme oxygenase 1 (HO1) in the LV myocardium after DAU treatment (Fig. 5A) which was completely prevented by the early DEX intervention in both study periods. On the other hand, the expression of HO1 was still significantly induced in the animals receiving delayed DEX intervention—especially at the end of treatment. The very similar findings were also obtained in the downstream members of heme degradation pathway—biliverdin reductase A (BVR A) and ferritin light chains (FTL) (Fig. 5B and C). Gene expression of 5-aminolevulinate synthase 1 (ALAS1) was regulated inversely due to DAU treatment and even this change was preventable by DEX administration (Fig. 5D). Thus, DAU-induced cardiotoxicity markedly up-regulated the key enzymes involved in heme breakdown, while it had opposite effect on the enzyme essential for heme synthesis in mitochondria (ALAS1). In addition, we found down-regulated expression of myoglobin in the DAU group (Fig. 6A)—one of the most abundant heme proteins in the heart. As the results did not suggest the regulation of HO1 pathway through Nrf2 and we did not see a distinct inflammatory reaction to imply the major regulatory role of NF␬B, we have focused on another potent transcriptional factor which is known to regulate HO1 expression—HIF1␣. We determined similar up-regulation of HIF1␣ gene expression due to DAU treatment along with its higher levels in the myocardial nuclear fraction. These alterations were prevented by DEX co-treatment, especially in the DD1 group in the FU period (Fig. 6B and C).

Fig. 9. Analysis of common deletions in mitochondrial DNA in the left ventricular myocardium. The long-range PCR analysis of the mtDNA segments between 5487–14,946 bp was performed and the PCR products were separated on 1% agarose gels. Only the bands corresponding with the length of amplified mtDNA segment were detected in all groups irrespectively to the treatments without any apparent difference in the signal intensity. No PCR products of significantly lower length were found in this study which suggests lack of deletion mutations including so called common deletions. “C” (control; CTR), “D” (daunorubicin; DAU), “1” (early dexrazoxane intervention; DD1 ) and “7” (delayed dexrazoxane intervention; DD7 ), “GR” (GeneRuler 1 kb DNA Ladder). Ethidium bromide was used for the detection of the PCR products.

3.6. DEX-induced prevention of mitochondrial damage The measurement of citrate synthase activity in the LV myocardium suggested a significant DAU-induced loss of mitochondrial content which was completely prevented by the early DEX intervention, whereas the delayed one was less effective (Fig. 7A). Further analyses showed that the early DEX administration also prevented DAU-induced decrease of relative abundance of mtDNA in the FU period (Fig. 7B), which was not the case of delayed dosage schedule. Since mtDNA copy number is regulated by mitochondrial transcription factor A (TFAM) we analyzed gene expression of this mitochondrial transcription factor to reveal that DEX co-treatment prevented severe TFAM down-regulation due to DAU treatment (Fig. 7C). In line with this we detected significant down-regulation of the expression of mitochondrial DNA-encoded subunits of complex I and IV (ND4 and COX1, Fig. 8A and B) in the DAU group, but the same was found in nuclear DNA-encoded subunits of these complexes (NDUFS2 and COX4, Fig. 8C and D). These changes were partially (the DD7 group) to completely (the DD1 group) prevented by DEX co-treatment. Similar trend was observed in gene expression of mitochondrial adenine nucleotide translocase type 1 (ANT1) and nuclear transcriptional factor 1 (NRF1), particularly in the FU period (Fig. 8E and F). However, analysis of the large rearrangements of the mtDNA did not confirm presence of common deletions or similar rearrangements after DAU treatment and DEX co-treatment had no impact on the outcomes (Fig. 9). 4. Discussion In the present study, we show that both early and delayed cardioprotective interventions with DEX can be effective in prevention of ANT-induced severe congestive heart failure. All DEX-treated animals survived till the scheduled end of the experiment (including the post-treatment follow up) without any apparent sign of blood congestion which contrasted with the animals receiving

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ANT alone. Hence, both treatment schedules offered certain benefit which is in line with the outcomes of clinical trials (Swain et al., 1997a,b). However, the degree of cardioprotection afforded by both strategies differed considerably. While the early DEX intervention offered very effective (almost complete) and long-lasting cardioprotection from ANT-induced cardiac dysfunction, this was not the case of the delayed one as here a significant drop of the LV systolic function was documented using echocardiography as well as LV catheterization examination. Quite surprisingly, both treatments showed comparable results in protection from diastolic dysfunction which may suggest that this type of cardiac dysfunction need not be always antecedent to the systolic one in chronic ANT cardiotoxicity as it has been proposed previously (Menna et al., 2012). The marked difference between outcomes of both cardioprotective interventions did not concern only parameters of cardiac function, but it was also obvious from cardiac morphology analysis as well as from determination of the biomarker of cardiac damage. Chronic DAU treatment induced typical degenerative changes in the LV myocardium which has been described previously with significant cumulative dose of different ANTs in patients as well as in different laboratory animals (Billingham et al., 1978; Herman and Ferrans, 1998; Van Vleet et al., 1980). The follow up of these changes suggested partial progression of the more advanced cardiomyocyte degeneration to the cell death along with the onset of the healing process represented mostly by replacement fibrosis. Importantly, early DEX intervention was apparently very effective in prevention of these morphological alterations as the myocardium morphology was almost normal and this assumption was further strengthened by the examination at the end of FU period. However, quite different morphological picture was found when delayed DEX intervention was employed. Here, the six DEX-free cycles of DAU administration were apparently responsible for development of myocardial degenerative changes, while subsequent DEX co-treatment prevented their further progression and allowed onset of healing process which continued in the FU period. This may explain why the severity of morphological changes was lower than in unprotected animals. Cardiac troponins has been repeatedly shown as quite sensitive biomarkers of cardiac damage in ANT cardiotoxicity (Adamcova et al., 2005; Herman et al., 1998; O’Brien et al., 1997) and the present study confirms that this is particularly true for high sensitive assays which have become available recently. This examination well documented the cumulative damage which happened to the myocardium with increasing cumulative dose of ANT and the FU data suggested that this is not finished with the last ANT dose. Of note, while the early DEX intervention prevented any raise of the cardiac damage biomarker, the delayed intervention just prevented its further progression since the DEX administration began. Hence, it is obvious that the delayed DEX intervention can only reduce further progression of either subclinical or clinical cardiac damage induced by previous ANT exposure. This is in line with the prevailing concept that each ANT cycle induces certain largely irreversible silent damage which accumulates cycle-by-cycle and manifests itself after overcoming the functional reserves of the heart and compensatory mechanisms (Aapro et al., 2011; Ewer and Ewer, 2010; Menna et al., 2012). In this context, delayed cardioprotection may indeed limit incidence of severe heart failure and cardiomyopathy associated with higher cumulative doses of ANTs, but it allows an induction of more subtle damage to the heart which may negatively affect long-term cardiovascular health of the cancer survivors. Considering the cumulative dose of DAU in this study, it should be noted that the rabbits are relatively sensitive to ANT cardiomyopathy development and hence, the cardiac damage found in our study is likely more severe than that found in patients with currently recommended delayed DEX intervention. Nevertheless, our data contributes to the previous concerns and discussion

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regarding the optimal schedule of cardioprotective intervention with DEX (Ewer and Speyer, 2006). Currently recommended delayed intervention with DEX replaced the early one in the last decade after the finding of the significant difference in the objective response rate of breast cancer between DEX and placebo co-treated patients in a 088001 trial (Swain et al., 1997b). However, no other oncological parameters (including time to disease progression or survival) were affected in this study and the similar trial no. 088006 (Swain et al., 1997a) with the same design showed no impact of DEX on any of the oncologic parameters. Moreover, no such effect has ever been reported from clinical trials before or after this study and latest meta-analysis of all randomized clinical trials with DEX has concluded that there is no evidence for significant effect of DEX on anticancer effect of ANTbased chemotherapy (van Dalen et al., 2011). Furthermore, DEX has been shown to have no impact on ANT pharmacokinetics in both experimental and clinical settings (Cvetkovic and Scott, 2005). In addition, while a study suggested that DEX could enhance risk of secondary malignancies (Tebbi et al., 2007), design of the study was a matter of discussion (Hellmann, 2007; Lipshultz et al., 2007) and a subsequent trial designed specifically for testing this hypothesis did not support this finding (Barry et al., 2008). Hence, in the light of these data, the rationale for delayed cardioprotective intervention may be inadequate from cardiovascular safety point of view and it may deserve reconsideration. The present study as well as virtually all experimental and clinical studies performed so far demonstrated the remarkably effective cardioprotective action of DEX in chronic ANT cardiotoxicity settings (Sterba et al., 2013; van Dalen et al., 2011). Hence, DEX may therefore be understood as a unique key to the enigma of anthracycline cardiotoxicity and effective prevention. Furthermore, the partial cardioprotection offered by the delayed DEX intervention may help to identify the molecular events associated with the effective cardioprotection and progression of ANT cardiotoxicity. The traditional concept perceives DEX as a pro-drug metabolized inside the myocytes to iron chelating agent (ADR-925) which prevents catalytic role of free iron in the formation of toxic hydroxyl radicals with subsequent oxidative damage to the heart (Hasinoff and Herman, 2007). In the present study, we found particular induction of oxidative stress in the FU period where significant and consistent impact of both cardioprotective interventions on the evaluated parameters was recognizable. However, while there was apparent difference in the cardiac phenotype between the DEX cotreated groups and DAU-only group at the end of treatment period, no significant and consistent differences were found in oxidative stress parameters or Nrf2 pathway. This may imply that oxidative stress rather followed the DAU-induced cardiac injury instead of being antecedent. These findings are in line with our earlier study (Popelova et al., 2009) and together they can argue against the leading hypothesis explaining remarkable cardioprotective effects of DEX by effective protection from direct ANT-induced oxidative injury. Interestingly, DEX had more apparent effects on myocardial lipoperoxidation and GSSG raise in the post-treatment follow up, which suggests that this later oxidative stress might be a consequence of the toxic injury induced before. Redox cycling of ANT molecule is unlikely to be responsible for this observation, nevertheless, our study offers alternative mechanisms including advancing mitochondrial damage, induction of expression of ROS generating NOX enzymes (NOX2 and NOX4) or/and downregulation of several antioxidant enzymes dealing with the free radicals, particularly in the mitochondria (MnSOD and PRDX3). The NOX enzymes as well as down-regulated MnSOD have been previously suggested to be responsible for ANT cardiotoxicity development (Wojnowski et al., 2005; Yen et al., 1999) and the former have been also supported by decreased toxicity of DOX after

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repeated dosing in NOX2 KO mice (Zhao et al., 2010). However, further studies are required to reveal whether DEX can directly interfere with the expression or activity of these targets or whether these are rather consequences of ANT-induce cardiac injury and dysfunction promoting its further progression. The present data do not support the traditional concept of iron chelation-mediated prevention of ANT-induced cardiac oxidative damage as the pivotal mechanism of DEX-afforded cardioprotection. It should be noted that we and others have failed to obtain the same or better degree of cardioprotection with stronger and more selective intracellular iron chelators as reviewed previously (Sterba et al., 2013). Furthermore, the active metabolite of DEX (ADR-925), is a EDTA-like metal chelator which forms complexes with iron, but the resulting chelate is still redox active and it may participate in the ROS production (Diop et al., 2000; Thomas et al., 1993) which might be a theoretical argument undermining the prevailing hypothesis. Furthermore, while an incubation of the cardiomyocytes with ADR925 has been demonstrated to induce intracellular iron chelation (Hasinoff, 2002), it has been found no protection against DOX toxicity (Hasinoff et al., 2003). Moreover, as stated before, virtually all classic antioxidants failed to show effective cardioprotection in the chronic ANT cardiotoxicity setting (Dresdale et al., 1982; Legha et al., 1982; Myers et al., 1983; Sterba et al., 2013). Interestingly, we found that chronic ANT treatment induces expression of HO1, BVR A and FTL which was partially to completely prevented by DEX co-treatment. This is likely an endogenous protective response of the heart towards the cardiac toxicity and damage which does not appear to be mediated by Nrf2 as the master regulator of antioxidant response, but instead our data suggests potential connection to HIF1␣ signaling. Interestingly, recent in vitro study has shown that DEX induces HIF1␣ expression in cardiomyocytes (Spagnuolo et al., 2011). Activation of HO1 pathway decreases availability of potentially redox active heme in the cell and our data also suggest decreased synthesis of this prosthetic group in mitochondria both which may be an attempt to deal with the oxidative stress associated with advancing myocardial damage. In the present study, we found that DAU cardiotoxicity is associated with down-regulation of expression of myoglobin at the mRNA level, while the same change at the protein level was reported previously (Sterba et al., 2011). The stressinduced activation of HO1 and BLVR pathway is known to increase endogenous antioxidant bilirubin and CO, which may attempt to counteract the oxidative stress and mitochondrial damage apparently induced by the mechanisms mentioned above during the progression of cardiac damage and dysfunction. Increased expression of ferritin due to ANT treatment is likely a consequence of the HO1 pathway activation to allow cardiomyocytes to deal with free iron released from heme to isolate it from participation on oxidative stress. Further studies employing genetically modified organisms could shed more light on the particular role of HO1 pathway in the chronic ANT cardiotoxicity development and potential therapeutic value of its pharmacological modulation. One of the interesting hypotheses explaining the chronic ANT cardiotoxicity development suggests that ANTs induce oxidative stress particularly in the mitochondria which results into formation of common deletions in mtDNA with subsequent impairment of expression of mtDNA-encoded subunits of respiratory chain (Lebrecht et al., 2003; Wallace, 2007). This could explain the certain delay between the exposure of the heart to ANTs and clinical manifestation of the cardiotoxicity as well as the impairment of mitochondrial bioenergetics and oxidative stress reported previously (Wallace, 2003). In the present study, we found that DEX protects the LV mitochondria from ANT toxicity especially when administered with every single ANT dose. However, the rather late change in mtDNA/nDNA ratio induced by ANT suggests that mtDNA need not be the primal target for the ANT toxicity.

Furthermore, a lack of ANT-induced common deletion in mtDNA as well as other gross rearrangements in the analyzed mtDNA segment argues against the above described mechanisms of cardioprotective effects of DEX in the early onset form of chronic ANT cardiotoxicity, while we cannot exclude that it might be more important for the delayed onset ones. In contrast to the previous finding (Lebrecht et al., 2003, 2007), we found marked down-regulation of expression of mitochondrial proteins encoded by both nuclear and mitochondrial genome, which further supports above given assumption. The global down-regulation of mitochondrial proteins in ANT cardiotoxicity settings seems to be orchestrated and this could be executed through suppressed mitochondrial biogenesis pathway as reported previously (Jirkovsky et al., 2012; Suliman et al., 2007; Zhang et al., 2012). Of particular note, the recent study by Zhang et al. (2012) showed that topoisomerase II␤ genetic deletion can effectively prevent cardiotoxicity induced by repeated DOX administration to mice via prevention of mitochondrial biogenesis disruption. In this context, it should be noted that DEX is a catalytic inhibitor of this enzyme and in vitro studies by the same authors showed that DEX treatment may decrease topoisomerase II␤ proteins levels via ubiquitin-proteasome system (Lyu et al., 2007). In addition, a very close DEX derivative (ICRF-161), which is free of topoisomerase inhibitory effect, has been found unable to protect rats from chronic ANT cardiotoxicity, although it has a similar pharmacokinetic profile as DEX and can also be similarly activated to the metal chelating metabolite (Martin et al., 2009). 5. Conclusion In conclusion, this study highlights the superiority of the early cardioprotective intervention with DEX over the currently recommended delayed administration from the ANT cumulative dose of 300 mg/m2 . While both schedules can be effective in preventing of ANT-induced severe congestive heart failure and premature deaths, only the early DEX intervention was found able to as nearly completely prevent LV dysfunction, myocardial morphological changes and mitochondrial damage. In addition, our data do not support the traditional hypothesis that this effect is based and directly dependent on the protection from ANT-induced oxidative stress or common deletions in mitochondrial genome. Instead, it is suggested that the effects of DEX on recently proposed topoisomerase II␤-mitochondrial biogenesis pathway may deserve further study. Altogether, the present data show that DEX can be an extremely effective cardioprotectant against chronic ANT cardiotoxicity; however, the current recommendation of delayed cardioprotection strategy may deserve reconsideration to exploit the full cardioprotective potential of the drug. Conflict of interest statement None declare. Acknowledgment We thank Mrs. Klára Lindrová and Jitka Pohorská for excellent technical assistance during the experiments and help of Generi Biotech (Czech Republic) with the analysis of mtDNA deletions. We also thank Mr. Matthew Spreadbury for reading of the manuscript and correction of English. This study was supported by the grant of Czech Science Foundation [13-15008S] and the Charles University research program PRVOUK [P37/5].

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2013.06.012.

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