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TOXICOLOGY
t
rba
im
nek
ová Hrochl
Department of Pharmacology (O.P., M.., M.H., V.G.), Department of Histology and Embryology (Y.M., I.G.), Department of Physiology (M.A.), Faculty of Medicine in Hradec Králové, and Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové (T..), Charles University in Prague, Hradec Králové, Czech Republic
Received February 5, 2008; accepted April 22, 2008.
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Abstract |
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). However, their clinical potential is considerably hampered by the risk of severe dose-dependent cumulative cardiotoxicity, which manifests months or years after the completion of chemotherapy (Minotti et al., 2004). These chronic and delayed types of cardiotoxicity are characterized by continuous and irreversible myofibrillar loss, cytoplasmic vacuolization, and degeneration of cardiomyocytes that can ultimately result into dilated cardiomyopathy and congestive heart failure (Herman et al., 1997).
Despite a number of theories proposed (Minotti et al., 2004; Chen et al., 2007), the precise molecular basis of this phenomenon still remains elusive. The prevailing hypothesis emphasizes the iron-catalyzed formation of reactive oxygen species (ROS) (Keizer et al., 1990). It is known that anthracyclines chelate free or loosely bound iron within the cardiomyocytes to form anthracycline-Fe3+ complexes, which can undergo cascade of reactions resulting in a production of extremely reactive and toxic hydroxyl radicals. In addition, anthracycline molecule can induce the production of superoxide radicals via redox cycling of the quinone/semiquinone ring of its aglycone. The superoxide produced through this route can dismutate to hydrogen peroxide, which may in turn enter the iron-catalyzed Haber-Weiss reaction, resulting again in an overproduction of hydroxyl radicals (Keizer et al., 1990). These can attack and damage all biomolecules (lipids, proteins, and nucleic acids) within their vicinity and thus promote the death of cardiac cells either by apoptosis or necrosis.
A number of interventions have been proposed to prevent the anthracycline-induced ROS formation (Saad et al., 2001; Oliveira et al., 2004; Bast et al., 2007). Nevertheless, so far, dexrazoxane (ICRF-187) is the only agent, which is clearly able to protect the myocardium from anthracycline-induced toxicity both in experimental and clinical settings (Wouters et al., 2005). Dexrazoxane is a prodrug, which is enzymatically hydrolyzed (inside the cardiomyocytes) to its active metal-chelating metabolite, ADR-925. This metabolite is believed to be responsible for the cardioprotective effects by displacing iron from its complex with anthracyclines and/or via chelation of intracellular labile iron pool (Hasinoff et al., 1998). However, dexrazoxane may potentiate the myelotoxicity of anthracyclines, which together with its high cost limit its wider use in clinical practice (van Dalen et al., 2006).
Deferiprone (Fig. 1, L1) is the first orally active iron chelator introduced into the clinical practice for the treatment of iron-overloaded patients (Kontoghiorghes et al., 2004). It is a synthetic bidentate chelator with a small molecular weight, binding specifically ferric iron in a 3:1 ratio. Under physiological conditions, it has a favorable lipophilicity and thus can readily enter the cardiomyocytes to reach therapeutic levels (Glickstein et al., 2006). It has been also shown to efficiently bind labile cellular iron, both free as well as accumulated, within mitochondria and lysosomes (Glickstein et al., 2006). Using an in vitro model of iron overload, L1 has been reported to significantly attenuate ROS formation within mitochondria and restore contractility impaired by iron loading (Link et al., 1996). L1 given orally on a daily basis has been also shown to effectively reduce myocardial iron burden and improve ventricular function in iron-overloaded patients with β-thalassemia (Anderson et al., 2002).
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). This effect has been attributed to its iron-chelating properties, which allow displacing the iron from its complexes with anthracyclines and/or reduce the availability of free iron for catalysis of the Haber-Weiss reaction. Moreover, a recent study performed on the isolated spontaneously beating atria further confirmed the cardioprotective potential of L1 against acute anthracycline cardiotoxicity (Xu et al., 2006). To summarize, several lines of evidence indicated that L1, already a clinically approved drug, might be a promising and readily available option to increase the cardiac safety of cancer patients undergoing treatment with anthracyclines. The present investigation was undertaken to further the development of this drug as a potential cardioprotectant. Two main aims of this study were 1) to preliminarily assess whether deferiprone has any potential to blunt the anticancer efficacy of anthracyclines (as this could preclude practical utility of this approach) and 2) to examine the cardioprotective potential of this drug using a clinically relevant model of chronic anthracycline cardiotoxicity, which was previously validated with dexrazoxane.
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Materials and Methods |
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Cardioprotection Studies in Rabbits
Animals. Forty-two adult Chinchilla male rabbits of an average body weight of 3.44 ± 0.03 kg at the beginning of the experiment were housed under a 12-h light cycle and constant temperature and humidity. The animals had free access to tap water and a standard laboratory pellet diet. Before experimental procedures, the animals were fasted overnight. Administration of drugs, blood sampling, and noninvasive measurements during the study were carried out under ketamine (50 mg/kg, i.m.; Narketan inj., Vétoquinol AG, Switzerland) and midazolam (1.25 mg/kg, i.m.; Midazolam Torrex; Torrex Chiesi Pharma GmbH, Vienna, Austria) anesthesia. Pentobarbital (30 mg/kg i.v.; Sigma-Aldrich) was used for anesthesia during final invasive hemodynamic measurements and for an overdose of animals at the end of the experiment. All experiments were approved and supervised by the Ethical Committee of Charles University (Prague, Czech Republic) and the Faculty of Medicine (Hradec Králové, Czech Republic) and were in accordance Institute of Laboratory Animal Resources (1996).
Experimental Design. All substances were administered once a week for 10 weeks. The study was carried out with five groups of animals: 1) control group—animals were receiving saline (1 ml/kg i.v., n = 8; Natrium Chloratum, Biotika, Slovakia); 2) DAU group—animals were injected with DAU (3 mg/kg i.v., n = 11; Daunoblastina; Pharmacia Italia S.p.A., Nerviano, Italy) in a validated schedule for induction of chronic anthracycline cardiomyopathy (Simunek et al., 2004); 3) L1 group—animals received L1 (50 mg/kg p.o., in 0.5% carboxymethylcellulose, n = 6; kindly provided by ApoPharma Inc.); 4) L1 10 + DAU group (n = 8)—this group received L1 (10 mg/kg p.o., in 0.5% carboxymethylcellulose) 45 min before each DAU administration (3 mg/kg i.v.); and 5) L1 50 + DAU group (n = 9)—L1 (50 mg/kg p.o.) was administered before DAU in the same schematic as in the previous combination group.
Body weight was recorded weekly, whereas mortality, general appearance, and behavior were observed daily. Noninvasive echocardiographic measurements were performed at the beginning of the study and later in weeks 8, 9, and 10 and, finally, at the end of the experiment (5–7 days after the last administration of drugs). Plasma for cardiac troponin T (cTnT) determination was sampled before the first, fifth, eighth, and tenth administration and at the end of the study. Standard biochemical and hematological parameters were determined from blood sampled from the ear artery before the first and fifth administrations and at the end of the study. The experiment was terminated 5 to 7 days after the last administration when final invasive hemodynamic measurements were performed. Thereafter, the animals had been overdosed with pentobarbital, and an autopsy had followed. The heart of each animal was rapidly excised, washed in ice-cold saline, and briefly retrogradely perfused with cold saline through the aorta. Tissue blocks of the transversely sectioned left and right cardiac ventricles underwent histological examination. The rest of the left ventricle was snap-frozen in liquid nitrogen and kept frozen at –80°C until further analyzed.
Noninvasive Cardiac Function Measurements. Echocardiographic examination of the LV systolic function was carried out using a Vivid 4 echocardiograph (GE Medical Systems Ultrasound; GE Healthcare, Chalfont St. Giles, UK) equipped with a 10-MHz probe. The LV long axis view was obtained through the left parasternal approach, and a guided M-mode measurement at the tips of the mitral valve was performed. The LV fraction shortening (LVFS) was calculated from the LV end-diastolic (LVED) and end-systolic diameters (LVES) determined from at least four heart cycles in each M-mode examination as follows:
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Invasive Hemodynamic Measurements. In pentobarbital anesthesia, the left carotid artery was prepared, and a Micro-Tip Pressure Catheter (2.3F Nylon; ADInstruments, Australia) was introduced into the left heart ventricle. After a 10-min equilibration period, the measurement of the following parameters was performed; the maximum of the first derivative of the LV pressure rise in the isovolumic phase of the systole (dP/dtmax) and the minimum of the first derivative of the LV pressure decline in the isovolumic phase of diastole (dP/dtmin) as well as the heart rate. For the arterial blood pressure measurement, a polyethylene cannula, filled in with heparinized (10 IU/ml) saline, was inserted into the right femoral artery. The ADI PowerLab/8SP (ADInstruments Pty Ltd., Castle Hill, Australia) with appropriate transducers and the Chart software 5.4.2 were used for pressure measurements, obtaining from derivatives and recordings.
Cardiac Troponin T Determination. cTnT, as a selective and sensitive marker of heart injury, was determined in heparinized plasma using an Elecsys Troponin T STAT Immunoassay (Roche Diagnostics, Basel, Switzerland) and an Elecsys 2010 (Roche Diagnostics) immunoassay analyzer with the detection limit of 0.010 ng/ml. The values below this detection limit were considered to be zero.
Determination of Total Malondialdehyde in Myocardial Samples. The samples of the LV myocardium were pulverized under liquid nitrogen. Radioimmunoprecipitation assay buffer (500 µl) was added to the myocardial samples (
70 mg), and the mixture was homogenized and vortexed. After centrifugation (3000 rpm, 10 min, 4°C), the supernatant was removed, and 250 µl of supernatant was taken and analyzed according to the Pilz et al. (2000) with minor modifications. In brief, 50 µl of NaOH (6 M) were added to the taken amount of supernatant, and after vortexing, the solution was kept at 60°C for 30 min. The samples were then cooled on ice, and 125 µl of perchloric acid [35% (v/v)] was added. After centrifugation (13,000 rpm, 10 min, 4°C), 250 µl of supernatant was taken, and derivatization was performed using 25 µl of 5 mM 2,4-dinitrophenylhydrazine. After 10 min in the dark, the solution (30 µl) was analyzed using an high-performance liquid chromatography system (Shimadzu, Kyoto, Japan) with UV detection: column—EC Nucleosil 100–5 C18, 4.6 x 125 mm heated on 30°C; mobile phase—acetonitrile/water/acetic acid: 380/620/2 (v/v/v), pH 3.4; flow rate—1.0 ml/min, UV detector set on 310 nm.
Histological Examination. Tissue blocks of the transversely sectioned left and right cardiac ventricles were fixed for 3 days by immersion in 4% neutral formaldehyde. Paraffin sections (6 µm thick) were stained with hematoxylin-eosin and Masson's blue trichrome. Photomicrographs were made with a Cybernetics software version 4.51 (Laboratory Imaging, Prague, Czech Republic).
Biochemical and Hematological Analyses. Standard biochemical parameters were determined in plasma/serum using an automatic analyzer (Hitachi 737; Hitachi, Tokyo, Japan) at the Institute of Clinical Biochemistry and Diagnostics, University Teaching Hospital (Hradec Králové, Czech Republic); hematological parameters were measured using an automatic analyzer Coulter T890 (Beckman Coulter, Fullerton, CA) at the Institute of Clinical Hematology, University Teaching Hospital. Content of iron in the left ventricular myocardium samples was determined at the Institute of Clinical Biochemistry and Diagnostics using graphite furnace atomic absorption spectrometry (Solaar 959; Thermo Fisher Scientific, Waltham, MA) as described previously (Simnek et al., 2005b). The results are expressed as micromole per gram of dry tissue.
Data Analysis. Statistical software SigmaStat 3.5 (SPSS Inc., Chicago, IL) and STATISTICA Cz (StatSoft, Tulsa, OK) were used in this study. All data are expressed as mean ± S.E.M. Significances of the differences were determined using one-way ANOVA unpaired test (comparison between groups) or paired t test (comparison with the initial value within each group). For exploratory data analysis (Principal Component Analysis, Hierarchical Tree Clustering), data from all parameters (general toxicity, cardiovascular, biochemical, and hematological parameters) were employed.
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Results |
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Determination of Malondialdehyde in Myocardial Samples. Malondialdehyde (MDA) was used as a marker of lipid peroxidation in the LV myocardium. As shown in Fig. 5, the MDA content was significantly increased in the DAU group. Although the MDA concentration was somewhat lower when L1 (10 mg/kg) was coadministered with DAU, the change did not reach the statistical significance. The escalation of the chelator dose did not improve the results, and MDA levels remained significantly higher than in controls and close to the DAU group.
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Invasive Hemodynamic Measurements. Invasively determined indexes of both the LV contractility (dP/dtmax) and relaxation (dP/dtmin) were significantly lower in the DAU than in the control group (Fig. 7). Concurrent administration of L1 in both doses resulted in similar outcomes with regard to both LV contractility and relaxation; no statistical differences were observed between these groups. A similar trend was determined in arterial blood pressure and heart rate values (Table 1).
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Cardiac Troponin T Determination. Ten weeks of repeated administration of DAU led to progressive and significant elevation of cardiac troponin T levels, starting with the 8th week (Fig. 8). Correspondingly, cotreatment with either dose of L1 induced very similar changes in plasma concentrations of cTnT as in the DAU group.
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With regard to myocardial iron content, no significant changes between the daunorubicin and control group were detected (2.2 ± 0.2 and 2.5 ± 0.4 µmol/g, respectively). However, significantly higher values were found in the L1-treated (50 mg/kg) and in the L1-(50 mg/kg) and DAU-cotreated animals (4.2 ± 0.7 and 4.9 ± 0.5 µmol/g, respectively, p 
0.05), whereas only insignificant difference was determined in the group cotreated with the lower dose of the chelator (3.4 ± 0.3 µmol/g).
Exploratory Data Analysis. Hierarchical tree clustering (Fig. 10A) and principal component analysis (Fig. 10B) are two independent exploratory multivariate statistical methods, which were used to identify the natural grouping of 42 individual animals in this study with respect to all evaluated parameters [general toxicity, cardiovascular, biochemical, and hematological parameters (25 variables)]. Both exploratory data analyses grouped all examined objects into two well separated clusters. In the first cluster (I), all animals from the control and L1 groups were found, whereas the individuals from the other groups (DAU, L1 10+DAU, and L1 50+DAU) were arranged into the second cluster (II).
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Discussion |
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; Xu et al., 2006) have strongly suggested that L1, a novel oral iron chelator, could be a rational and promising alternative to dexrazoxane in cardioprotective indications. To further explore therapeutic potential of L1, there was a need to eliminate a possibility that it can attenuate the antiproliferative efficacy of anthracyclines. Hence, using a leukemic cell line, we have demonstrated that this does not take place. In contrast, L1 alone was capable of significantly reducing proliferation of leukemic cells in the concentrations, which might be achieved in vivo. Furthermore, when HL-60 cells were treated with DAU (
IC50) together with different concentrations of L1, it was obvious that the chelator did not possess any potential to blunt the antiproliferative effect. Instead, in higher doses of L1, the summation of antiproliferative effects of both compounds was observed. These outcomes are in good agreement with other reports describing antiproliferative effects of L1 (Yasumoto et al., 2004) and other iron-chelating compounds (Yu et al., 2006). Several lines of evidences suggest that inhibition of ribonucleotide reductase can be involved in this matter (Green et al., 2001), although other mechanisms have been also proposed. Different iron chelators have been shown to cause cell cycle arrest and apoptosis and antimetastatic and antiangiogenic effects (Richardson, 2005). Using L1 and HL-60 leukemic cell line, it has been shown that chelator treatment induces apoptosis via both intrinsic (mitochondria-mediated) and extrinsic pathways (Yasumoto et al., 2004). Furthermore, recently, it has been reported that L1 anticancer effects can be related to the deregulation of the polyamine metabolism. It has been suggested L1 anticancer effects are associated with induction of spermidine/spermine N1-acetyltransferase pathway leading to higher putrescine levels, resulting in the cell cycle arrest in G2-M phase (Lescoat et al., 2007).
In the major part of the study, we aimed to assess whether L1 cotreatment can overcome or significantly diminish chronic anthracycline cardiotoxicity. For this purpose, a clinically relevant dexrazoxane-validated rabbit model of chronic anthracycline cardiotoxicity was used (Simnek et al., 2004).
L1 was administered orally in two doses of 10 and 50 mg/kg, 45 min before each DAU injection. It is surprising that, in both doses, L1 was unable to reduce anthracycline-induced mortality. Moreover, the higher L1 dose (50 mg/kg) actually led to earlier deaths, which can probably be explained by the occurrence of extracardiac toxicity, the nature of which could not be reliably identified in this study. Most importantly, we have also clearly demonstrated that L1 is not able to protect the myocardium against either anthracycline-induced oxidative stress or LV cardiac damage and heart failure. Using a validated high-performance liquid chromatography method, we have found marked elevation of MDA (a standard marker of lipoperoxidation) following DAU administration, which is in line with numerous reports published so far (Lebrecht et al., 2007). However, we have surprisingly failed to detect a significant impact of L1 on this parameter in either dose. This evidently contradicts the results provided in previous reports (Barnabé et al., 2002; Xu et al., 2006). Nevertheless, both the latter studies could have certain limitations with respect to translatability of their results to the clinically relevant situation. In the first study, although L1 has been shown to reduce the anthracycline-induced ROS production using electron paramagnetic resonance spectroscopy, this was not followed within the biological system and particularly not in the chronic setting. In second case, myocardial injury was induced with an acute exposure of the isolated atria (which are not the main target for anthracyclines) to relative high and clinically unachievable doxorubicin concentration (30 µM).
The inability of L1 to overcome DAU-induced oxidative stress was further supported by the outcomes of functional LV examination, performed using both echocardiography and LV catheterization. Furthermore, L1 exerted no effect on DAU-induced plasma concentrations cTnT, which is a very sensitive and selective biochemical marker of cardiac injury (Sterba et al., 2007b). The good agreement of these results was further supported by similar findings from the histopathological examination of the myocardium. On the other hand, repeated administration of L1 alone (in the higher dose of 50 mg/kg) was well tolerated, and no abnormalities were found in the most of the parameters evaluated, which corresponds with a preclinical investigation of L1 performed on a number of species (Porter et al., 1990). The empiric assumptions given above were further supported by two independent exploratory data analyses. These have clearly revealed that L1 had shown no clear tendency to affect the general toxicity, cardiovascular, hematological, and plasma biochemistry parameters. The only exception was found in the case of the higher myocardial iron content in the animals treated or cotreated with the higher dose of the chelator. This finding was rather surprising in the light of the proved efficacy of this compound to mobilize iron from the myocardium of iron-overloaded animals and patients upon regular daily treatment (Anderson et al., 2002; Wood et al., 2006). Furthermore, the biological half-life (approximately 2 h) of L1 excludes any potential direct effect of the compound on this parameter a week after its last dose. Hence, one can only reasonably explain these findings like a certain adaptive reaction of myocardium on the repeated intermittent iron chelation. Most importantly, these changes have not been associated with increased oxidative stress or other correlating myocardial disturbances, which indicates that this compensatory reaction was instead within a physiological range.
The results of this study strongly suggest that oral treatment with L1 has no beneficial effect on chronic anthracycline cardiotoxicity, which certainly raises several questions. One may speculate that the failure of L1 could have been associated with its poor or erratic bioavailability. However, it should be noted that L1 is characterized with very good oral bioavailability; it is fairly well absorbed from the gastrointestinal tract in humans as well as in rabbits (Fredenburg et al., 1993). Furthermore, the doses and timing of administration were designed according to the pharmacokinetic study performed in rabbits. It is noteworthy that L1 concentrations affording significant protection against anthracycline cardiotoxicity in vitro are detectable in rabbit plasma, even more than 12 h after its oral administration (Fredenburg et al., 1993). Furthermore, potential pharmacokinetic interactions also deserve consideration in such experiments. However, in this particular case, the experimental design and especially the pharmacokinetic characteristics of both studied compounds (Yokel et al., 1995; Danesi et al., 2002) make this possibility unlikely. It should be noted that neither of these drugs is extensively bound to plasma proteins. Moreover, both drugs are known to have different routes of elimination. Whereas L1 is mainly excreted by kidney to the urine, daunorubicin is predominantly excreted by liver to the stool. In addition, both drugs also do not share the major metabolic pathways, and neither of these agents is known as an important substrate for cytochrome P450. Furthermore, direct pharmacokinetic interaction at the cardiomyocyte level is also unlikely, as this would be recognizable in the previous in vitro and ex vivo results.
Similar to dexrazoxane, L1 is able to quickly enter cardiomyocytes (Glickstein et al., 2006) and efficiently remove iron from its complex with doxorubicin (Barnabé et al., 2002). Thus, it should be able to protect myocytes against anthracycline-induced injury. The discrepancy between this assumption and the outcomes of the present study prompts us to compare L1 with other iron chelators studied in a similar setting so far [e.g., lipophilic, cell permeable, and specific chelator of iron, deferasirox (ICL670), studied on the same model as L1 lacked any protective effects]. It is interesting that this occurred in spite of the fact that ICL670 quickly and efficiently removed Fe3+ from its complex with doxorubicin, rapidly entered myocytes, and displaced iron from an intracellular iron-calcein complex (Hasinoff et al., 2003). This suggests that the mere ability of an agent to chelate iron need not be a sole determinant of its protective action. Difference in cardioprotective properties could be accounted for different ratios in which chelators are capable to bind ferric ions. L1 is a bidentate iron chelator, which forms a complex with iron in a ratio of 3:1. This might be a limitation from the viewpoint of protection, because the probability of insufficient occupation of all sites of ferric ions is higher. Furthermore, we cannot rule out that, although L1 entered the cardiac cells, it did not reach the desirable intracellular compartment and remained there for a sufficient time to induce effective cardioprotection.
Previously, the well known iron chelator deferoxamine had been shown to be ineffective against chronic anthracycline toxicity in spontaneously hypertensive rats (Herman et al., 1994). Nevertheless, this was well attributable to the hydrophilic nature of this drug, which hinders its penetration into the cardiomyocytes. On the other hand, using our in vivo rabbit model, we have recently shown that lipophilic aryolhydrazone iron chelators are able to protect against anthracycline cardiotoxicity in terms of overall mortality, functional parameters, and histopathology (Simunek et al., 2005a; Sterba et al., 2006, 2007a). However, at the same time, it was revealed that dexrazoxane is superior to aroylhydrazones, as the cardioprotection of the latter compounds—albeit significant—was always only partial. Moreover, dose escalations of all aroylhydrazones surprisingly resulted in the disappearance of protection, which was puzzling and unexpected. These results together with the findings from the present study indicate that the role of iron in anthracycline cardiotoxicity is not as trivial as originally supposed. For instance, a number of studies provided evidence for anthracycline-induced dysregulation of iron homeostasis with potentially serious consequences (Xu et al., 2005). Anthracyclines can perturb iron metabolism by interacting with various molecular targets, including iron regulatory proteins, ferritin, or transferrin receptor (Kwok and Richardson, 2003; Xu et al., 2008).
The observed lack of cardioprotective effect of L1 can also mean that iron-catalyzed formation of ROS is not the pivotal and ultimate executor responsible for chronic anthracycline cardiotoxicity. ROS can also be produced without presence of iron, and they might be rather important as the triggering factor for a number of successive molecular and cellular events. In addition, anthracyclines and their metabolites have been reported to induce a number of distinct cellular effects, which do not appear to be ROS-mediated (Menna et al., 2007). For example, anthracyclines have been demonstrated to induce a number of perturbations in cellular calcium homeostasis (Simunek et al., 2005b; Wallace, 2007). Unlike in the case of selective iron chelator L1, it can not be excluded that dexrazoxane may also chelate calcium, which can potentially account for the difference in cardioprotective effects of both chelators.
This study also points out on difficulties with the translation of in vitro and ex vivo cardioprotection results into the chronic in vivo settings, reflecting more closely the anthracycline cardiotoxicity seen in clinics. It is interesting that a similar scenario took place with a number of antioxidants (e.g., vitamins A and E and acetylcysteine) (Dresdale et al., 1982; Legha et al., 1982; Myers et al., 1983).
In conclusion, this study revealed that, despite the promising results obtained previously in vitro/ex vivo, iron chelation with L1 was unable to protect the myocardium against lipoperoxidation, cardiomyopathy, and heart failure induced by repeated administration of DAU to rabbits. Together with our previous findings, this study strongly suggests that the role of iron and its chelation in anthracycline cardiotoxicity is probably not as trivial as originally believed and/or other mechanisms unrelated to iron-catalyzed ROS production are involved in this pathology.
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Acknowledgements |
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ková for myocardial iron content determination, Ludmila Lat
nová for skillful technical assistance, and Dr. John Connelly (ApoPharma Inc.) for kindly providing a drug substance of deferiprone. |
Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DAU, daunorubicin; L1, deferiprone; ROS, reactive oxygen species; ICRF-187, dexrazoxane; cTnT, concentration of troponin T; ICL670, deferasirox; MDA, malondialdehyde; LV, left ventricular; FS, fractional shortening; ANOVA, analysis of variance; ADR-925, N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)glycine].
Address correspondence to: Dr. Olga Popelová, Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Králové, imkova 870, 500 38 Hradec Králové, Czech Republic. E-mail: popelovao{at}lfhk.cuni.cz
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), L1 (
), DAU (
), L1 10+DAU (+), and L1 50+DAU (
).