Dexrazoxane-afforded protection against chronic anthracycline cardiotoxicity in vivo: effective rescue of cardiomyocytes from apoptotic cell death.

BACKGROUND: Dexrazoxane (DEX, ICRF-187) is the only clinically approved cardioprotectant against anthracycline cardiotoxicity. It has been traditionally postulated to undergo hydrolysis to iron-chelating agent ADR-925 and to prevent anthracycline-induced oxidative stress, progressive cardiomyocyte degeneration and subsequent non-programmed cell death. However, the additional capability of DEX to protect cardiomyocytes from apoptosis has remained unsubstantiated under clinically relevant in vivo conditions. METHODS: Chronic anthracycline cardiotoxicity was induced in rabbits by repeated daunorubicin (DAU) administrations (3 mg kg(-1) weekly for 10 weeks). Cardiomyocyte apoptosis was evaluated using TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) assay and activities of caspases 3/7, 8, 9 and 12. Lipoperoxidation was assayed using HPLC determination of myocardial malondialdehyde and 4-hydroxynonenal immunodetection. RESULTS: Dexrazoxane (60 mg kg(-1)) co-treatment was capable of overcoming DAU-induced mortality, left ventricular dysfunction, profound structural damage of the myocardium and release of cardiac troponin T and I to circulation. Moreover, for the first time, it has been shown that DEX affords significant and nearly complete cardioprotection against anthracycline-induced apoptosis in vivo and effectively suppresses the complex apoptotic signalling triggered by DAU. In individual animals, the severity of apoptotic parameters significantly correlated with cardiac function. However, this effective cardioprotection occurred without a significant decrease in anthracycline-induced lipoperoxidation. CONCLUSION: This study identifies inhibition of apoptosis as an important target for effective cardioprotection against chronic anthracycline cardiotoxicity and suggests that lipoperoxidation-independent mechanisms are involved in the cardioprotective action of DEX.

Anthracycline toxicity to cardiomyocytes or cancer cells is differently affected by iron chelation with salicylaldehyde isonicotinoyl hydrazone.

BACKGROUND AND PURPOSE: The clinical utility of anthracycline antineoplastic drugs is limited by the risk of cardiotoxicity, which has been traditionally attributed to iron-mediated production of reactive oxygen species (ROS). EXPERIMENTAL APPROACH: The aims of this study were to examine the strongly lipophilic iron chelator, salicylaldehyde isonicotinoyl hydrazone (SIH), for its ability to protect rat isolated cardiomyocytes against the toxicity of daunorubicin (DAU) and to investigate the effects of SIH on DAU-induced inhibition of proliferation in a leukaemic cell line. Cell toxicity was measured by release of lactate dehydrogenase and staining with Hoechst 33342 or propidium iodide and lipid peroxidation by malonaldehyde formation. KEY RESULTS: SIH fully protected cardiomyocytes against model oxidative injury induced by hydrogen peroxide exposure. SIH also significantly but only partially and with no apparent dose-dependency, reduced DAU-induced cardiomyocyte death. However, the observed protection was not accompanied by decreased lipid peroxidation. In the HL-60 acute promyelocytic leukaemia cell line, SIH did not blunt the antiproliferative efficacy of DAU. Instead, at concentrations that reduced DAU toxicity to cardiomyocytes, SIH enhanced the tumoricidal action of DAU. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that iron is most likely involved in anthracycline cardiotoxicity and that iron chelation has protective potential, but apparently through mechanism(s) other than by inhibition of ROS-induced injury. In addition to cardioprotection, iron chelation may have considerable potential to improve the therapeutic action of anthracyclines by enhancing their anticancer efficiency and this potential warrants further investigation.

Evaluation of ECG time intervals in a rabbit model of anthracycline-induced cardiomyopathy: a useful tool for assessment of cardioprotective agents.

The aim of this study was to analyze the ECG time intervals in the course of the development of chronic anthracycline cardiomyopathy in rabbits. Furthermore, this approach was employed to study the effects of a model cardioprotective drug (dexrazoxane) and two novel iron chelating compounds--salicylaldehyde isonicotinoyl hydrazone (SIH) and pyridoxal 2-chlorobenzoyl hydrazone (o-108). Repeated daunorubicin administration induced a significant and progressive prolongation of the QRS complex commencing with the eighth week of administration. At the end of the study, we identified a significant correlation between QRS duration and the contractility index dP/dt(max) (r = -0.81; P<0.001) as well as with the plasma concentrations of cardiac troponin T (r = 0.78; P<0.001). In contrast, no alterations in ECG time intervals were revealed in the groups co-treated with either dexrazoxane or both novel cardioprotective drugs (SIH, o-108). Hence, in this study, the QRS duration is for the first time shown as a parameter suitable for the non-invasive evaluation of the anthracycline cardiotoxicity and cardioprotective effects of both well established and investigated drugs. Moreover, our results strongly suggest that novel iron chelators (SIH and o-108) merit further study as promising cardioprotective drugs against anthracycline cardiotoxicity.

Early detection of anthracycline cardiotoxicity in a rabbit model: left ventricle filling pattern versus troponin T determination.

Anthracycline cardiotoxicity represents a serious risk of anticancer chemotherapy. The aim of the present pilot study was to compare the potential of both the left ventricular (LV) filling pattern evaluation and cardiac troponin T (cTnT) plasma levels determination for the early detection of daunorubicin-induced cardiotoxicity in rabbits. The echocardiographic measurements of transmitral LV inflow as well as cTnT determinations were performed weekly for 10 weeks in daunorubicin (3 mg/kg weekly) and control groups (n=5, each). Surprisingly, no significant changes in LV-filling pattern were observed through the study, most likely due to the xylazine-containing anesthesia, necessary for appropriate resolving of the E and A waves. In contrast to the echographic measurement, the dP/dt(min) index obtained invasively at the end of the study revealed a significant impairment in LV relaxation, which was further supported by observed disturbances in myocardial collagen content and calcium homeostasis. However, at the same time cTnT plasma levels were progressively rising in the daunorubicin-treated animals from the fifth week (0.024+/-0.008 microg/l) until the end of the experiment (0.186+/-0.055 microg/l). Therefore, in contrast to complicated non-invasive evaluation of diastolic function, cTnT is shown to be an early and sensitive marker of anthracycline-induced cardiotoxicity in the rabbit model.

Iron is not involved in oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin.

BACKGROUND AND PURPOSE: The anticancer drugs doxorubicin and bleomycin are well-known for their oxidative stress-mediated side effects in heart and lung, respectively. It is frequently suggested that iron is involved in doxorubicin and bleomycin toxicity. We set out to elucidate whether iron chelation prevents the oxidative stress-mediated toxicity of doxorubicin and bleomycin and whether it affects their antiproliferative/proapoptotic effects. EXPERIMENTAL APPROACH: Cell culture experiments were performed in A549 cells. Formation of hydroxyl radicals was measured in vitro by electron paramagnetic resonance (EPR). We investigated interactions between five iron chelators and the oxidative stress-inducing agents (doxorubicin, bleomycin and H(2)O(2)) by quantifying oxidative stress and cellular damage as TBARS formation, glutathione (GSH) consumption and lactic dehydrogenase (LDH) leakage. The antitumour/proapoptotic effects of doxorubicin and bleomycin were assessed by cell proliferation and caspase-3 activity assay. KEY RESULTS: All the tested chelators, except for monohydroxyethylrutoside (monoHER), prevented hydroxyl radical formation induced by H(2)O(2)/Fe(2+) in EPR studies. However, only salicylaldehyde isonicotinoyl hydrazone and deferoxamine protected intact A549 cells against H(2)O(2)/Fe(2+). Conversely, the chelators that decreased doxorubicin and bleomycin-induced oxidative stress and cellular damage (dexrazoxane, monoHER) were not able to protect against H(2)O(2)/Fe(2+). CONCLUSIONS AND IMPLICATIONS: We have shown that the ability to chelate iron as such is not the sole determinant of a compound protecting against doxorubicin or bleomycin-induced cytotoxicity. Our data challenge the putative role of iron and hydroxyl radicals in the oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin and have implications for the development of new compounds to protects against this toxicity.

Safety and tolerability of repeated administration of pyridoxal 2-chlorobenzoyl hydrazone in rabbits.

Recently, pyridoxal 2-chlorobenzoyl hydrazone (o-108) has been identified as an effective iron chelator [Link et al., Blood 2003; 101: 4172-79]. Since chronic treatment would be necessary in its potential indications, in the present study, the safety and tolerability of this agent after repeated administration was determined. Three doses of o-108 (25, 50, 100 mg/kg, in 10% Cremophor EL) were administered intraperitoneally, once weekly, for 10 weeks to three groups (n=5 each) of Chinchilla male rabbits. The effects on biochemical, haematological and cardiovascular parameters were examined during the experiment; histopathological examination was performed at the end of the experiment. Results were compared with control (saline 2 mL/kg, n=11) and vehicle groups (10% Cremophor EL, 2 mL/kg, n=12). No premature deaths occurred; the well-being of animals was evidenced by their body weight gain, although lower gain was observed with the highest dose (100 mg/kg). Significant elevations of cardiac troponin T plasma concentrations were observed with the highest dose of o-108, but no abnormalities were found in the cardiovascular function and only minor and inconsistent changes in haematological and biochemical parameters were observed. Histopathological examinations of selected organs revealed only weak and reversible changes through all studied groups. Thus, the data from this study suggest that o-108 remains a promising drug from the standpoint of the possibility of its repeated administration and warrants further investigation.

Study of daunorubicin cardiotoxicity prevention with pyridoxal isonicotinoyl hydrazone in rabbits.

Risk of cardiotoxicity is the most serious drawback of the clinical usefulness of anthracycline antineoplastic antibiotics, which however, remain among the most powerful and widely employed anticancer drugs. In this study we have used daunorubicin-induced cardiomyopathy in rabbits as a model to investigate possible cardioprotective effects of pyridoxal isonicotinoyl hydrazone (PIH)-a principal representative of a novel group of aroylhydrazone iron chelators. Three groups of animals were used: a control group (n=11; i.v. saline), daunorubicin-treated animals (n=11; 3mg/kg, i.v.), and animals pretreated with PIH (n=9, 25 mg/kg, i.p.) 60 min before daunorubicin administration. All substances were administered once weekly for 10 weeks. Repeated administration of daunorubicin caused premature death in four animals and induced conspicuous histopathological changes in the myocardium, progressive and significant impairment of systolic heart function (a decrease in left ventricular dP/dt(max), ejection fraction, an increase in the pre-ejection period/left ventricular ejection time index), and a gradual increase in cardiac troponin T plasma concentrations. On the contrary, all the PIH-treated animals have survived all daunorubicin applications. Furthermore, in this group, the daunorubicin-induced cardiac changes were in most functional, biochemical as well as morphological parameters less pronounced than in the group receiving daunorubicin alone. Hence, PIH and other aroylhydrazones merit further investigation as potentially protective agents against anthracycline-induced cardiotoxicity.

Protein profiling in daunorubicin-induced cardiomyopathy.

The aim of this paper was to study the protein remodelling of the left ventricle following repeated administration of either daunorubicin (DNR) or DNR in combination with the cardioprotective agent dexrazoxane (DXZ). The experiment was carried out on three groups of Chinchilla male rabbits: 1. DNR (3 mg/kg i.v.), 2. DNR (3 mg/kg i.v.) + DXZ (60 mg/kg i.p.), and 3. the control group (saline 1 ml/kg i.v. in the same schedule). The drugs were given once weekly, max. 10 administrations. Protein fractions were isolated by stepwise extraction from the samples of the left ventricle. In the DNR-group, the concentrations of both, metabolic and contractile proteins were significantly reduced, while the amount of collagen was significantly higher in comparison with the control group. In the group treated with DNR and DXZ, the concentrations of individual protein fractions (except metabolic proteins) were comparable to those of the control group, which confirms a significant cardioprotective effect of DXZ. The changes of protein profiling corresponded to functional examination of both cardiac parameters (EF, dP/dt(max), PEP: LVET index) and histological examination. These data should be used in further studies dealing with evaluation of cardiotoxic and, possibly, cardioprotective effects of new drugs.

Cardiac troponin T following repeated administration of pyridoxal isonicotinoyl hydrazone in rabbits.

Pyridoxal isonicotinoyl hydrazone (PIH) is a new tridentate Fe-chelating agent that should be very promising in many pathological states resulting from both an iron-overload and formation of free radicals. The aim of our study was to investigate the effect of PIH on the cardiovascular system focusing to the regulatory protein -- cardiac troponin T (cTnT). The study was carried out in two groups of Chinchilla male rabbits: 1) PIH (50 mg/kg dissolved in 10 % Cremophor i.p., once a week, 10 administrations, n=8) and 2) Cremophor (2 ml/kg i.p. in the same schedule, n=7). Plasma concentrations of cTnT (as a marker of myocardial damage) were measured using a commercial kit (Roche). cTnT was within the physiological range (i.e. < 0.1 microg/l) during the whole experiment in the Cremophor group. In the PIH group, the cTnT levels were not significantly increased when compared with the control group or with the initial values (except with those before the 5th administration). Furthermore, we analyzed the cytosolic and myofibrillar fraction of cTnT in the left ventricular myocardium. Using SDS-PAGE and Western blot we resolved three isoforms. The profiling of TnT did not differ significantly between the PIH-treated group and the Cremophor-treated group. Our data concerning cTnT support the opinion that the possible cardiotoxicity of PIH is very low.

Anthracycline-induced cardiotoxicity.

Anthracycline antibiotics are among the most effective and widely used antineoplastic drugs. Their usefulness is limited by a cumulative dose-related cardiotoxicity, whose precise mechanisms are not clear as yet. The principal role is possibly exerted by free oxygen radicals generated by "redox-cycling" of anthracycline molecule and/or by the formation of anthracycline-ferric ion complexes. The iron catalyzes the hydroxyl radical production via Haber-Weiss reaction. The selective toxicity of ANT against cardiomyocytes results from high accumulation of ANT in cardiac tissue, appreciable production of oxygen radicals by mitochondria and relatively poor antioxidant defense systems. Other additional mechanisms of the anthracycline cardiotoxicity have been proposed--calcium overload, histamine release and impairment in autonomic regulation of heart function. The currently used methods for an early identification of anthracycline cardiotoxicity comprise ECG measurement, biochemical markers, functional measurement and morphologic examination. Among a plenty of studied cardioprotective agents only dexrazoxane (ICRF-187) has been approved for clinical use. Its protective effect likely consists in intracellular chelating of iron. However, in high doses dexrazoxane itself may cause myelotoxicity. This fact encourages investigation of new cardioprotectants with lower toxicity. Orally active iron chelators and flavonoids attract more attention. Modification of dosage schedule and synthesis of new anthracycline analogues may represent alternative approaches to mitigate anthracycline cardiotoxicity while preserving antitumour activity.