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Toxicology 289 (2011) 122– 131

Contents lists available at ScienceDirect

Toxicology

j ourna l ho me pag e: www.elsev ier .com/ locate / tox ico l

omparison of various iron chelators used in clinical practice as protectinggents against catecholamine-induced oxidative injury and cardiotoxicity

avlína Hasková, Lucie Koubková, Anna Vávrová, Eliska Macková, Katerina Hrusková,etra Kovaríková, Katerina Vávrová, Tomás Simunek ∗

harles University in Prague, Faculty of Pharmacy in Hradec Králové, Czech Republic

r t i c l e i n f o

rticle history:eceived 10 July 2011eceived in revised form 2 August 2011ccepted 4 August 2011vailable online 12 August 2011

eywords:atecholamine cardiotoxicity

ron chelationesferrioxamine (DFO)eferiprone (L1)eferasirox (ICL670A)

a b s t r a c t

Catecholamines are stress hormones and sympathetic neurotransmitters essential for control of cardiacfunction and metabolism. However, pathologically increased catecholamine levels may be cardiotoxicby mechanism that includes iron-catalyzed formation of reactive oxygen species. In this study, fiveiron chelators used in clinical practice were examined for their potential to protect cardiomyoblast-derived cell line H9c2 from the oxidative stress and toxicity induced by catecholamines epinephrineand isoprenaline and their oxidation products. Hydroxamate iron chelator desferrioxamine (DFO) signif-icantly reduced oxidation of catecholamines to more toxic products and abolished redox activity of thecatecholamine-iron complex at pH 7.4. However, due to its hydrophilicity and large molecule, DFO wasable to protects cells only at very high and clinically unachievable concentrations. Two newer chelators,deferiprone (L1) and deferasirox (ICL670A), showed much better protective potential and were effectiveat one or two orders of magnitude lower concentrations as compared to DFO that were within their clin-ically relevant plasma levels. Ethylenediaminetetraacetic acid (EDTA), dexrazoxane (ICRF-187, clinically

approved cardioprotective agent against anthracycline-induced cardiotoxicity) as well as selected betaadrenoreceptor antagonists and calcium channel blockers exerted no effect. Hence, results of the presentstudy indicate that small, lipophilic and iron-specific chelators L1 and ICL670A can provide significantprotection against the oxidative stress and cardiomyocyte damage exerted by catecholamines and/ortheir reactive oxidation intermediates. This potential new application of the clinically approved drugs L1

ther

and ICL670A warrants fur

. Introduction

Iron (Fe) is an essential element for all mammalian cells. Due tots optimal flexibility to serve as both electron donor and acceptor,e is the key component of various proteins critical in basic cellular

Abbreviations: DCF, 2′ ,7′-dichlorofluorescein; DFO, desferrioxamine; DMEM,ulbecco’s modified Eagle’s medium; DMSO, dimethylsulphoxide; D-RZX or ICRF-87, dexrazoxane; EC50, concentration leading to 50% protection from toxicity;DTA, ethylenediaminetetraacetic acid; EPI, epinephrine; FAC, ferric-ammoniumitrate; FAS, ferrous-diammonium sulphate; FDA„ Food and Drug Administration;e, iron; HBS, HEPES-buffered saline; H2DCF-DA, 2′ ,7′-dichlorodihydrofluorescein-iacetate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid; IBE,

ron-binding equivalent; ICL670A, deferasirox; ISO, isoprenaline; L1 or CP-0, deferiprone; NR, neutral red; PBS, phosphate buffered saline; PCTH,-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone; PI, propidium iodide;OS, reactive oxygen species; SIH, salicylaldehyde isonicotinoyl hydrazone; TC50,oncentration inducing 50% viability loss compared to untreated control.∗ Corresponding author at: Charles University in Prague, Faculty of Pharmacy,epartment of Biochemical Sciences, Heyrovského 1203, 500 05 Hradec Králové,zech Republic. Tel.: +420 495 067 422; fax: +420 495 067 168.

E-mail addresses: Tomas.Simunek@faf.cuni.cz, simunekt@faf.cuni.czT. Simunek).

300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2011.08.006

investigation, preferably using more complex in vivo animal models.© 2011 Elsevier Ireland Ltd. All rights reserved.

processes such as oxygen transport and storage, electron transport,cellular respiration, DNA, RNA and protein synthesis, cellular prolif-eration and differentiation and regulation of gene expression (Lieuet al., 2001). However, unless appropriately shielded, this transitionmetal can be dangerous and toxic as it may significantly contributeto oxidative injury of various susceptible tissues, such as brain orheart (Halliwell and Gutteridge, 2007; Jomova and Valko, 2011;Kell, 2010; Valko et al., 2005). Transition metals including Fe accel-erate the Haber–Weiss reaction of superoxide radical (•O2

−) andhydrogen peroxide (H2O2). The Fe-catalyzed Haber–Weiss reac-tion consists of two parts: reduction of ferric ion (Fe3+) by •O2

−

to ferrous (Fe2+) which in turn reacts with H2O2 (Fenton reaction)yielding hydroxyl radicals (•OH) – the most reactive and toxic formof reactive oxygen species (ROS):

•O2− + Fe3+ → O2 + Fe2+

H2O2 + Fe2+ → OH− + •OH + Fe3+ (Fenton reaction)

In summary:

•O2− + H2O2 (Fe2+/Fe3+) → O2 + OH− + •OH

(Haber–Weiss reaction)

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Considering the propensity of free Fe to mediate oxidativenjury, cells are equipped with specific proteins designed for Fecquisition, transport and storage as well as with sophisticatedechanisms that maintain the intracellular labile and redox-active

e pool at an appropriate level. Nevertheless, due to the lack offfective body Fe excretion, excess of body Fe occurs in some dis-ases with increased Fe absorption and/or regular blood transfusionherapy, such as �-thalassemia major.

In addition, local Fe-mediated oxidative injury may occur evenithout systemic Fe overload. Reactive oxygen or nitrogen speciesay severely deregulate cellular Fe homeostasis (Mladenka et al.,

006). For example, high cellular levels of superoxide and peroxidepecies, as well as low pH (which occur during the acute myocardialnfarction), are able to release Fe from its storage proteins, increasehe cytosolic pool of labile Fe and thus promote the productionf deleterious ROS (Reif, 1992; Voogd et al., 1992). Furthermore,edox-active Fe may also promote formation of ROS within mito-hondria and lysosomes (Terman and Brunk, 2006) and thus closehe vicious cycle of propagation of oxidative injury.

Oxidative stress is a common denominator of a wide rangef pathological conditions such as diabetes, ischemia-reperfusionnjury of cardiac or brain tissues, cystic fibrosis, chronic inflamma-ory diseases or cancerogenesis (Halliwell and Gutteridge, 2007).owever, conventional treatment of these diseases still lacks these of antioxidants due to negative study outcomes. Nevertheless,fforts to reduce toxicity of oxidative stress have so far focusedrimarily on the use of ROS scavengers, rather than on actual pre-ention of ROS production. The latter can be achieved with Fehelators, i.e. agents that form redox-inactive complexes with Fend block the Fe-dependent production of •OH. Recent experi-ental evidence suggests that Fe chelation may efficiently reduce

he neuro- and cardio-toxicity induced by catecholamines (Ben-hachar et al., 1991; Haskova et al., 2011; Mladenka et al., 2009a).

Catecholamines are important neurotransmitters of centralnd sympathetic nervous system and regulators of myocardial

ig. 1. Chemical structures of the studied iron chelators: ethylenediaminetetraacetic acid

errioxamine (FO), deferiprone (L1) and its iron complex (L1-Fe), deferasirox (ICL670A) anroduct ADR925 and its iron complex (ADR925-Fe).

289 (2011) 122– 131 123

contractility and metabolism. However, an excessive release oradministration of catecholamines may induce neuro- and cardio-toxic effects. Catecholamines are known to participate in Parkinsondisease development (Sulzer, 2007) as well as to deteriorate mani-festation of ischemic heart disease, induce stress cardiomyopathy,cardiac arrhythmias and/or heart failure (Costa et al., 2011; Dhallaet al., 2010).

In certain pathophysiological conditions, the catecholamineconcentrations are markedly elevated and the plasma levels ofendogenous catecholamines and their metabolites vary over aseveral thousand-fold range (Goldstein et al., 2003). In pheochro-mocytoma patients, plasma concentrations of norepinephrine werereported as high as 57 �M (Gerlo and Sevens, 1994). In addition, iso-prenaline (ISO) has been frequently used to induce model cardiacinjury in experimental animals (Mladenka et al., 2009b; Rajaduraiand Prince, 2007). Remião et al. reported that 15 min after theintraperitoneal administration of 80 mg/kg ISO to rats, its concen-trations in the heart were between 100 and 200 �M (Remiao et al.,2002).

The mechanisms of catecholamine cardiotoxicity are notcompletely understood. Apart from excessive �-adrenoreceptorstimulation and/or induction of calcium overload of cardiac cells,increasing body of experimental evidence suggests that oxidativestress is involved in the catecholamine-induced cardiomyopathyand that not parent catecholamines per se, but rather their reac-tive oxidation intermediates are responsible for this toxicity (Costaet al., 2011; Dhalla et al., 2010).

To this end, five clinically approved Fe chelating agents (Fig. 1)were examined in this study and compared for their ability toprevent catecholamine autooxidation, and, most importantly, toprotect the rat ventricular cardiomyoblast-derived cell line H9c2

against the intracellular oxidative stress and cell death inducedby catecholamines epinephrine (EPI) and ISO and their oxidationproducts. Apart from the three drugs approved for treatment of Feoverload, namely, desferrioxamine (DFO), deferiprone (L1, CP-20),

(EDTA) and its iron complex (EDTA-Fe), desferrioxamine (DFO) and its iron complexd its iron complex (ICL670A-Fe), pro-chelator dexrazoxane (D-RZX), its hydrolysis

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as statistically significant. The chelator concentration inducing 50% viability loss ascompared to control (TC50) and the chelator concentration leading to 50% protec-tion from toxicity induced by catecholamines or their oxidation products (EC50)were calculated using CalcuSyn 2.0 software (Biosoft, Cambridge, U.K.).

Fig. 2. Assessment of the redox activities of the chelator-iron complexes. Effects ofchelators and their complexes with iron, alone or in combination with epinephrinepre-oxidized for 24 h in incubator at 37 ◦C (24h-oxEPI), on spontaneous ascorbate

24 P. Hasková et al. / Toxi

nd deferasirox (ICL670A), this study aimed to assess the potentialrotective effects of dexrazoxane (D-RZX, ICRF-187), an agent used

n clinical practice to prevent anthracycline-induced cardiotoxicitynd the unspecific metal chelator ethylenediaminetetraacetic acidEDTA). Furthermore, the effects of Fe chelators were comparedith two �-adrenoreceptor antagonists – propranol and carvedilol

nd two calcium channel blockers – nifedipine and amlodipine.

. Methods

.1. Chemicals

DFO and ICL670A were purified from commercial pharmaceutical preparationsf Novartis (Basel, Switzerland), L1 was a kind gift of ApoPharma (Weston, Canada)nd D-RZX was purchased from Chiron (Amsterdam, The Netherlands). EDTA, cat-cholamines – EPI and ISO, propranolol, carvedilol, nifedipine, amlodipine, variouse salts including ferric chloride (FeCl3), ferric-ammonium citrate (FAC), ferrous-iammonium sulphate (FAS), as well as other chemicals (e.g. constituents of variousuffers) were purchased from Sigma (Germany) or Penta (Czech Republic) and weref the highest available pharmaceutical or analytical grade. ADR925 was synthesizeds described previously and its identity and purity were confirmed using IR, 1H and3C NMR spectroscopy and HPLC with MS detection (Kovarikova et al., 2011).

.2. Ascorbate oxidation assay for the assessment of redox activities of chelates

Redox-active Fe ions markedly accelerate spontaneous oxidation of many sub-tances including ascorbic acid; therefore the spectrophotometric assay based oneasurement of oxidized ascorbate level has been used to examine the potential

ffects of Fe chelators (and formed Fe chelates) on the inhibition, preservation ortimulation of this reaction (Dean and Nicholson, 1994). This was also used to assesshe role of oxidized EPI in ascorbate oxidation induced by Fe. Ascorbic acid (50 �M)as dissolved in phosphate-citrate buffer (0.5 mM Na-citrate, 10 mM Na2HPO4; pH

.4) immediately prior to the experiment and incubated in the presence of 10 �MeCl3, a 50-fold molar excess of citrate (500 �M), and chelator in an iron bindingquivalent (IBE) ratio of 3. Ascorbate oxidation was determined as an absorbanceecrease, measured using a microplate spectrophotometer TECAN Infinite 200 MTECAN, Austria) at � = 265 nm at 60 min. Redox activity was expressed as a percent-ge of control, i.e. an ascorbate solution made up as above without FeCl3 (100%).

.3. Spectrophotometric determination of catecholamine autooxidation

Spontaneous EPI oxidation was measured using changes in absorption spectraver time (Haskova et al., 2011). 100 �M solutions of EPI, FAC, chelators (alone orn combinations) were prepared in ADS buffer (116 mM NaCl, 5.3 mM KCl, 1.2 mM

gSO4, 1.13 mM NaH2PO4, 1 mM CaCl2, 0.5 �M FAC, 20 mM HEPES; pH 7.4) andept in the dark at 37 ◦C throughout the experiment. EPI solutions were incubatedor up to 24 h alone or in combinations with FAC and/or chelator (each at 100 �Moncentration). Absorption spectra were scanned using a Helios � spectrophotome-er (ChromSpec, Czech Republic) at � = 200–800 nm following 0-, 6-, 12- and 24-hncubations. The EPI oxidation level was then expressed as an absorbance change at

= 295 nm (the wavelength with the highest spectral difference).

.4. Cell culture

The H9c2 cardiomyoblast line derived from embryonic rat heart tissue (Kimesnd Brandt, 1976) was obtained from the American Type Culture Collection (ATCC,.S.A.). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza,elgium) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin,nd 10 mM HEPES buffer (pH 7.4) in 75 cm2 tissue culture flasks. Incubations wereerformed at 37 ◦C in a humidified atmosphere of 5% CO2 in air. Cells were subcul-ured twice weekly, once they reached approximately 80–90% confluence.

24 h before experiments, the medium was changed to serum- and pyruvate-freeMEM (Sigma, Germany). Serum deprivation was used to stop cellular proliferation

o mimic the situation in post-mitotic cardiomyocytes (Simunek et al., 2008). Pyru-ate was removed from the medium since it is an antioxidant and may interfereith ROS-related toxicity. To dissolve lipophilic chelators, DMSO was used at final

oncentration of 0.1% and this concentration was used in all experimental groups.t this concentration, DMSO had no effect on cellular viability.

.5. Microscopic assessments of cellular morphology and viability

Changes in cellular morphology were assessed using an inverted epifluores-ence microscope Nikon Eclipse TS100 with 10–40× air objectives (Nikon, Japan)

nd a digital camera 1300Q (VDS Vosskühler, Germany). Furthermore, in selectedxperiments the cellular nuclei were stained with Hoechst 33342 and propidiumodide (PI; Molecular Probes, U.S.A.) for distinction of intact from apoptotic andecrotic cells. H9c2 cells seeded in 12-well plates at a density of 75,000 cells/wellere incubated with the parent or oxidized catecholamines and chelators (alone

289 (2011) 122– 131

or in combinations). After incubations with the test substances, cells were stainedwith 10 �g/ml of Hoechst 33342 and 1 �g/ml of PI for 20 min at room temperature.

2.6. Neutral red uptake assay

Cellular viability was determined using the cytotoxicity assay based on the abil-ity of viable cells to incorporate neutral red (NR). This weak cationic dye readilypenetrates intact cell membranes and accumulates in the lysosomes of viable cells(Repetto et al., 2008).

H9c2 cells seeded in a 96-well plate at a density 10,000 cells/well were incubatedwith the parent or oxidized catecholamines and chelators (alone or in combinations)for 24 h. Following the cell incubations with the tested substances, half of the vol-ume of medium from each well was removed, and the same volume of medium withNR was added (yielding a final NR concentration of 40 �g/ml). After 3 h at 37 ◦C, thesupernatant was discarded, and the cells were fixed in 0.5% formaldehyde supple-mented with 1% CaCl2 for 15 min. The cells were then twice washed with PBS andsolubilised with 1% acetic acid in 50% ethanol during 30 min of continuous agitation.The absorption of soluble NR was measured using a microplate spectrophotome-ter TECAN at � = 540 nm. The viability of experimental groups was expressed as apercentage of the untreated control (100%).

2.7. H2DCF-DA assay for the determination of cellular reactive oxygen speciesformation

To assess ROS generation inside H9c2 cells, measurement of 2′ ,7′-dichlorodihydrofluorescein-diacetate (H2DCF-DA) oxidation was used. Thisnonfluorescent reagent diffuses passively through the plasma membrane intothe cell, where the acetate groups are cleaved by intracellular esterases. Thecompound can then be oxidized by ROS formed within the cell (particularly byhydroxyl radicals – •OH) to form fluorescent DCF, while the fluorescence intensityis proportional to the ROS level (Wang and Joseph, 1999).

H9c2 cells seeded in a 96-well plate at a density of 10,000 cells/well weretwice washed by ADS buffer (116 mM NaCl, 5.3 mM KCl, 1.2 mM MgSO4, 1.13 mMNaH2PO4, 5 mM glucose, 1 mM CaCl2, 20 mM HEPES; pH 7.4) and loaded with 10 �MH2DCF-DA in ADS buffer. After 60-min incubation, the loading buffer was discarded,and cells were twice washed by ADS buffer and exposed to the oxidized cate-cholamines and chelators (alone or in combination) prepared in ADS buffer. Allsolutions were pre-warmed to 37 ◦C.

Fluorescence intensity was measured for 30 min at 37 ◦C using a microplatereader TECAN at �ex = 485 nm and �em = 525 nm. ROS formations in the experimentalgroups were expressed as a percentage of the untreated control (100%).

2.8. Data analysis

The statistical software SigmaStat for Windows 3.5 (SPSS, U.S.A.) was used inthis study. Data are presented as means ± S.E.M. of a given number of experiments.The Grubbs test was used for detection of outlier values. For multiple compar-isons either One Way ANOVA with Bonferroni post hoc analysis or One Way ANOVAon Ranks with Dunn’s post hoc analysis (data without normal distribution) wereused. For comparisons of two groups either Student’s t-test or the non-parametricMann–Whitney Rank Sum Test were used. P values of less than 0.05 were considered

oxidation determined as the decrease in UV absorbance at � = 265 nm following60 min incubations in buffered solution (pH 7.4). Chelators were prepared at three-fold concentrations of their iron binding equivalent (3x IBE). Data are presented asmeans ± S.E.M.; n = 4; statistical significance (ANOVA, P < 0.05): * vs. control, # vs.ferric ammonium citrate (FAC), � vs. 24h-oxEPI, $ vs. 24h-oxEPI with FAC.

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. Results

.1. Ascorbate oxidation assay for assessments of redox activitiesf chelator-Fe complexes

As seen in Fig. 2, addition of FAC significantly increased the spon-aneous oxidation of ascorbate. While the 24 h-preincubated EPIid not induce ascorbate oxidation when added alone, in a com-ination with FAC it promoted ascorbate oxidation more than FAClone. This ascorbate oxidation was completely prevented by DFO,1, ICL670A and ADR925. EDTA significantly reduced the ascorbate

xidation induced by combination of FAC with 24 h-preincubatedPI, but, on the contrary, it significantly increased the oxidationnduced by FAC alone. Pro-chelator D-RZX had no effect on ascor-ate oxidation.

ig. 3. Effect of iron chelators on spontaneous oxidation of epinephrine. (A–E) Time couEPI) alone or in combinations with ferric ammonium citrate (Fe) and/or various iron cheresented as means ± S.E.M.; n = 4; statistical significance (ANOVA, P < 0.05): * vs. epineph

289 (2011) 122– 131 125

3.2. Spectrophotometric determination of Fe-chelation effect onspontaneous and Fe-induced catecholamine oxidation

Fe ions markedly enhance the catecholamines’ oxidationresulting in formation of oxidation products; therefore the mea-surements of the changes in UV/VIS absorption spectra ofepinephrine have been used. Previously, it was established thatthe increase in absorbance at � = 295 nm was proportional to thedecrease in parent catecholamine concentration as determined byHPLC (Haskova et al., 2011).

As seen in Fig. 3, changes of absorbance at � = 295 nm were

followed in the time-course of 24-h incubations of EPI. Gradualincreases of absorbance were observed in time and these were sig-nificantly augmented by addition of FAC. Only EDTA and ICL670Asignificantly suppressed spontaneous oxidation of EPI at the end of

rses of absorbance changes at � = 295 nm during 24-h incubation of epinephrinelators (all 100 �M): (A) EDTA, (B) DFO, (C) L1, (D) ICL670A and (E) D-RZX. Data arerine.

126 P. Hasková et al. / Toxicology 289 (2011) 122– 131

Fig. 4. Effect of iron chelators on cellular oxidative stress induced by oxidized iso-prenaline. Cellular reactive oxygen species formation was determined in situ byH2DCF-DA assay following the 30-min incubation of H9c2 cardiomyoblast cell linewith tested substances or their combinations. After the intracellular acetate groupcleavage, increase in fluorescence intensity (�ex = 488 nm; �em = 520 nm) is propor-tional to probe oxidation by reactive oxygen species (particularly hydroxyl radicals)to green-fluorescent DCF. Intracellular fluorescence was expressed as a percentageof the untreated control group. Cells were incubated with various concentrations ofibn

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Fig. 5. Overview of the protocols for cytoprotection experiments using the H9c2cardiomyoblast cell line. Tested drug: iron chelator (CHEL), �-blocker (BB) or calciumchannel blocker (CCB) was added to cell-culture medium with catecholamine (CA– isoprenaline or epinephrine) which was either (A) freshly prepared or (B and C)24 h-preincubated at 37 ◦C. The tested potentially protective agent was added either(A and B) at the beginning of 24-h cellular experiments or (C) at the start of 24-hcatecholamine preincubation before the cellular experiments.

Fig. 6. Protection of H9c2 cardiomyoblast cell line against isoprenaline (ISO)- andepinephrine (EPI)-induced toxicities using various iron chelators. Cellular viabili-ties were determined by neutral red uptake assay and expressed as a percentageof the untreated control group. (A) Chelators were added to catecholamine freshlyprepared in cell-culture medium before the start of 24-h cellular experiments; (B)chelators were added immediately before cellular experiments to catecholaminespreincubated for 24 h in cell-culture medium; (C) chelators were preincubated for

ron chelators added immediately before cellular experiments to medium preincu-ated for 24 h with isoprenaline (24h-oxISO). Data are presented as means ± S.E.M.;

= 4; statistical significance (ANOVA, P < 0.05): * vs. control, # vs. isoprenaline group.

4-h incubation time (Fig. 3A and D); other chelators have shownrend towards the reduction of catecholamine oxidation, but thisid not reach the level of statistical significance. Of all the testedhelators, only DFO was able to significantly suppress the oxidationugmented by FAC (Fig. 3B).

.3. Determination of intracellular reactive oxygen speciesormation

As seen in Fig. 4, 60 �M concentration of 24 h-preincubatedSO significantly augmented ROS production inside the H9c2 cells

ithin the 30-min incubation as compared to control group. Co-ncubations with EDTA, DFO or D-RZX had no effect on DCFormation. On the contrary, the highest used concentrations of L1nd ICL670A (30 and 100 �M) suppressed this formation to levelshat did not differ significantly from untreated control group, whilehe 100 �M concentration of ICL670A significantly reduced the ROSormation also as compared to ISO group.

.4. Cellular toxicity studies using the H9c2 cardiomyoblast celline

The H9c2 rat cardiomyoblast-derived cell line was used forssessment of the potential cardioprotective effects of studied sub-tances against the toxicity exerted by catecholamines (ISO orPI) and their oxidation products. Cytotoxicity studies were basedn neutral red uptake assay and three different protocols shownn Fig. 5 were used. Firstly, the protective effects of Fe chelators1–3000 �M) were assayed in an experimental setup in which thehelator was co-incubated for 24 h with freshly prepared EPI orSO solutions at a concentration of 300 �M, which was previouslyhown to induce complete loss of cellular viability (Haskova et al.,011). As seen in Fig. 6A, DFO, L1 and ICL670 were able to partiallyrotect the cells in a concentration-dependent manner; the bestrotection was achieved with ICL670A, which significantly reducedytotoxic effect of ISO at 100 �M concentration (its upper solubilityimit); DFO showed significant reduction of ISO toxicity at higheroncentrations (300–3000 �M; EC50 = 577 �M for ISO-induced tox-city); L1 showed tendency to preserve cellular viability; however,his was not statistically significant.

Furthermore, the protective potential of Fe chelator was assayedgainst the toxicities induced by oxidized ISO and EPI (both at

0 �M concentrations) preincubated for 24 h prior to cellular expo-ure. Chelators were added to cell-culture medium either followinghe 24-h catecholamine oxidation, i.e. immediately before thetart of cellular experiments (Fig. 5B), or they were added to the

24 h together with catecholamines in cell-culture medium and then added to cells.(D) Concentration dependencies of chelators’ own toxicities to H9c2 cardiomy-oblast cell line following 24-h incubations. Data are presented as means ± S.E.M.;n = 4; Statistical significance (ANOVA, P < 0.05): * vs. control, # vs. correspondingcatecholamine.

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edium at the start of catecholamine preincubation (Fig. 5C).n either case, all Fe chelators (with exception of D-RZX) wereble to significantly protect the cells (Fig. 6B and C). The best

rotective efficiency was achieved with L1 (significant toxicityeduction at 100–300 �M concentrations; EC50 = 73 �M and 79 �Mor ISO- and EPI-induced toxicity, respectively, in case of chela-or addition after catecholamine preincubation and EC50 = 62.5 �M

ig. 7. Cellular morphology and nuclear epifluorescence co-stainings with Hoechst 333ontrol medium, (B) 24 h-preincubated epinephrine, or 24 h-preincubated epinephrine in

cale bars represent 50 �m.

289 (2011) 122– 131 127

and 59.5 �M for ISO- and EPI-induced toxicity, respectively, incase of chelator preincubation together with catecholamine) andICL670 (significant toxicity reduction at 10–30 �M concentra-

tions; EC50 = 5.5 �M and 7.0 �M for ISO- and EPI-induced toxicity,respectively, in case of chelator addition after catecholamine prein-cubation and EC50 = 6.0 �M and 9.5 �M for ISO- and EPI-inducedtoxicity, respectively, in case of chelator preincubation together

42 and propidium iodide. H9c2 cardiomyoblasts were incubated for 24 h with (A)combination with iron chelators (C) EDTA, (D) DFO, (E) L1, (F) ICL670A or (G) D-RZX.

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ith catecholamine). As seen in Fig. 6B and C, co-incubations of theells with L1 resulted in the best (i.e., nearly complete) restorationf cellular viability. Although to a lesser extent, DFO was also ableo effectively protect the cells, particularly in case of chelator prein-ubation together with catecholamine (EC50 = 377 �M and 600 �Mor ISO- and EPI-induced toxicity respectively). EDTA protectivefficiency did not reach 50%.

Own cytotoxicities of the studied Fe chelators following their4-h incubations with H9c2 cells are shown in Fig. 6D. Significanteductions of cellular viability were observed at concentrations of000 �M of D-RZX, 300 �M of EDTA (TC50 = 667 �M), DFO and L1,nd 10 �M of ICL670A.

In addition, fluorescent microscopy was used for assessmentsf cellular morphology. Incubations of H9c2 cells with oxidized EPIesulted in pronounced morphological signs of toxicity: peripheralembrane blebbing, loss of the spindle-like cell shape, rounding

p of cells, nuclear condensation and eventually complete loss ofellular viability followed by formation of cell debris. At the endf 24-h cellular incubation periods, nuclei were stained with twouorescent intercalating dyes – Hoechst 33342 and PI. As seen

n Fig. 7, while the control H9c2 cells displayed the vast major-ty of dark blue nuclei stained only by cell membrane-permeableoechst 33342 (Fig. 7A), the EPI-treated cells were shrunken and

tained by cell membrane-impermeable red PI, indicative of cellu-

ar necrosis (Fig. 7B). Protection by Fe chelators (except for D-RZX)gainst the EPI cytotoxicity was evident from the nuclear stain-ng with Hoechst 33342 and PI as well as preservation of cellular

orphology (Fig. 7C–F).

ig. 8. Lack of protection of H9c2 cardiomyoblast cell line against isoprenaline (ISO)-nd epinephrine (EPI)-induced toxicities using �-adrenergic receptor blockers (BBs)r calcium channel blockers (CCBs). Cellular viabilities were determined by neutraled uptake assay and expressed as a percentage of the untreated control group.A) BBs or CCBs were added to catecholamines freshly prepared in cell-culture

edium before the start of 24-h cellular experiments; (B) BBs or CCBs were addedmmediately before cellular experiments to catecholamines preincubated for 24 hn cell-culture medium; (C) concentration dependencies BBs or CCBs’ own toxicitieso H9c2 cardiomyoblast cell line following 24-h incubations. Data are presented as

eans ± S.E.M.; n = 4; statistical significance (ANOVA, P < 0.05): * vs. control, # vs.orresponding catecholamine.

289 (2011) 122– 131

In addition to various Fe chelators, two �-blockers (propra-nolol and carvedilol) and two calcium channel blockers (nifedipineand amlodipine) were also examined for potential cytoprotectiveeffects against cardiotoxicity induced by EPI, ISO and their oxi-dation products. As seen in Fig. 8A and B, none of these studieddrugs manifested ability to significantly protect the H9c2 cells inany experimental protocol. These agents were assayed in wideconcentration range: from nontoxic 0.1 �M to 30 �M, a dose thatdisplayed own significant cytotoxic effects (Fig. 8C). Carvedilol andamlodipine reached their median lethal concentration (TC50 = 13.2and 9.2 �M, respectively).

4. Discussion

Although chronic Fe overload causes damage to many organs,accumulation of cardiac Fe is the leading cause of death (Hershkoet al., 2005; Kremastinos et al., 2010; Wood, 2009). Low molecu-lar weight non-transferrin-bound (labile) Fe is oxidatively active,can enter the heart through nonspecific, poorly regulated cationchannels and result in cardiac Fe overload (Wood et al., 2005).Once labile Fe is increased in the cardiomyocyte, it destabilizeslysosomal, mitochondrial and sarcoplasmic membranes, furtherincreasing myocyte oxidative stress (Gujja et al., 2010); calcium,sodium, and potassium ion channels are disrupted causing con-duction/repolarization disturbances, arrhythmias, and diastolicand systolic dysfunction. Excessive Fe can even modify geneexpression, stimulate fibrosis, and trigger cardiomyocyte apoptosisand/or necrosis that progressively lead to heart failure and death(Kremastinos et al., 2010; Wood, 2009). Fe chelating agents havebeen used in clinical practice to promote Fe excretion in overloadedpatients (Hershko et al., 2005; Kalinowski and Richardson, 2005;Olivieri and Brittenham, 1997). This pharmacological Fe chelationhas been particularly beneficial due to the reduction of cardiac mor-tality (Wood, 2008, 2009). However, apart from correction of bodyFe accumulation, recent experimental evidence suggests that theconcept of Fe chelation may be also useful to prevent Fe-mediatedaggravation of oxidative stress, which can occur locally even in thepresence of normal Fe stores (Bendova et al., 2010; Galey, 2001;Simunek et al., 2005a).

Low concentrations of endogenous catecholamines stimulatethe heart by promoting Ca2+ movements. However, increased cate-cholamine levels are associated with cardiac injury and may triggeracute myocardial infarction. It has been suggested that this cat-echolamine cardiotoxicity is – at least partially – associated withredox cycling of catecholamines, their conversion to aminochromesand other highly reactive oxidation products and induction ofoxidative stress (Adameova et al., 2009; Costa et al., 2011; Dhallaet al., 2010). We have previously demonstrated significantly highercytotoxicity of oxidized catecholamines than of the parent com-pounds, apparently through the induction of caspase-independentcell death (Haskova et al., 2011). Furthermore, two experimental Fechelators, namely 2-pyridylcarboxaldehyde 2-thiophenecarboxylhydrazone (PCTH) and salicylaldehyde isonicotinoyl hydrazone(SIH), were recently shown as highly effective in reducing thecatecholamine-induced cardiotoxicity. Both PCTH and SIH wereshown to protect the cardiomyocyte-derived H9c2 cells in vitro(Haskova et al., 2011; Mladenka et al., 2009a). PCTH was also testedin vivo, where it significantly protected rats against the mortal-ity and cardiotoxicity induced by ISO administration (Mladenkaet al., 2009a). Experiments with SIH have revealed that the sig-nificant protective action of Fe chelation with SIH was associatedwith at least two distinct effects: (i) slowing down the progres-

sive catecholamine oxidation to reactive toxic intermediates and(ii) reduction of toxicity of the already formed oxidation products(Haskova et al., 2011). Unfortunately, both PCTH and SIH are exper-imental agents and particularly the lack of patent protection of SIH

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akes this otherwise highly efficient cardioprotective agent withow toxicity unattractive for pharmaceutical industry. To this end,n the present study we aimed at assessment of protective potentialf five chelating agents that have been already approved for vari-us applications of human medicine (Kalinowski and Richardson,005).

EDTA is used as a non-specific metal chelating agent in chemicalndustry or laboratory applications. Regarding its use in medicine,he FDA-labeled indications for systemic use include emergencyreatment of hypercalcemia and digitalis toxicity. In alternative

edicine, “chelation therapy” with EDTA is an unproven, yet pop-lar and widely used procedure for prevention or treatment ofardiovascular disease, in particular atherosclerosis (Seely et al.,005). In our experiments, EDTA showed potential to retard theatecholamine autooxidation and has shown small (albeit signifi-ant) ability to preserve the viability of H9c2 cells incubated withSO oxidation products. On the other hand, EDTA failed to suppressntracellular ROS formation. This may have been caused by lack ofigand selectivity for Fe and/or redox activity of the EDTA-Fe chelateGraf et al., 1984), which was in our study clearly demonstratedy the ascorbate oxidation assay. Hence, our results indicate very

imited potential of EDTA in prevention of catecholamine-inducedardiomyocyte oxidative injury.

Since 1963, microbial siderophore DFO was the first chelatinggent used in clinical practice to remove excess Fe from the body.ecause of its high molecular weight (657 g/mol), DFO mesylate

s not orally bioavailable and it is administered by subcutaneousnjection. Hence, despite its efficacy, patient compliance is poor.robably due to its long-time commercial availability, DFO hasained biggest attention also in experimental studies examin-ng potential of Fe chelation in pathological states with normale stores. Although DFO has been shown to significantly reduceschemia–reperfusion injury in various tissues including the heartBolli et al., 1987), in numerous other studies it failed to afford sig-ificant protection (Reddy et al., 1991; Watanabe et al., 1993). Thelausible explanation of limited DFO efficiency is its poor ability tonter the cardiac cells. In our study, DFO was able to retard the cat-cholamine autooxidation, formed redox-inactive iron complexesnd showed some potential to prevent the catecholamine toxic-ty towards the H9c2 cardiomyoblast cells. DFO was particularlyffective in the experiments with parent catecholamines, where itsfficiency could be largely attributed to the prevention of forma-ion of catecholamine oxidation products. When the chelator wasdded only after the 24-h preincubation period of catecholamines,t was considerably less efficient. Furthermore, it should be notedhat the effective DFO concentrations (≥300 �M) largely exceedhe highest DFO plasma levels seen in clinical settings, which are15 �M (Summers et al., 1979). At the latter concentration, no sig-ificant cytoprotection occurred and DFO also failed to reduce the

SO-induced intracellular ROS formation.Hydroxypyridinone Fe chelator deferiprone (L1, CP20) was the

rst orally active agent available in clinical practice. In contrast toFO, L1 is a small lipophilic molecule; it penetrates well into var-

ous tissues and is able to remove excess Fe from cardiac tissueHoffbrand et al., 2003). So far, only limited number of studies haseen published examining the antioxidant potential of L1 in situ-tions without Fe overload: L1 protected neuronal cells as well asbroblasts against H2O2-induced cell death in vitro (Lim et al., 2008;olina-Holgado et al., 2008) and it also protected rat hearts against

schemia-reperfusion injury (van der Kraaij et al., 1989). L1 alsoxerted protection of isolated cardiomyocytes against the toxicityf anthracycline doxorubicin (Barnabe et al., 2002), but was ineffec-

ive in an animal experiment (Popelova et al., 2008). In this study,1 was highly efficient in the prevention of toxicity of oxidized EPInd ISO, where it was able to restore cellular viability from 0% to80% of the untreated control cells (100%). Its efficiency towards

289 (2011) 122– 131 129

the parent catecholamines was only slight and insignificant, whichis in agreement with the observed lack of significant inhibition ofcatecholamine autooxidation. Using the ascorbate oxidation assay,L1 was clearly shown to form redox-inactive L1-Fe complexes aswell as to diminish the redox activity of the complex of oxidizedEPI and Fe. Apparently due to its good permeation into the cells, L1also significantly reduced the intracellular ROS formation inducedby oxidized catecholamine to the level that did not differ signifi-cantly from the control cells. Of note, the concentrations at whichL1 was protective (100–300 �M) are only slightly above the plasmalevels of this agent seen in clinical practice (Morales et al., 2009).

Deferasirox (ICL670A) is the newest clinically approved orallyactive Fe chelator. It is a synthetic triazole chelator that has beenalso shown capable to remove cardiac Fe and attenuate myocar-dial oxidative stress in the state of Fe overload (Al-Rousan et al.,2009). In the present study, ICL670A was able to significantly retardthe spontaneous catecholamine oxidation, blocked the intracellularROS formation and exerted significant protection against the cel-lular toxicity of catecholamines and their oxidation products withthe lowest effective concentrations of all the tested agents (3 �M).These protective concentrations were well within clinically rele-vant plasma concentrations (Chirnomas et al., 2009). On the otherhand, ICL670A displayed also the highest own toxicity, with slight– yet significant – viability reduction of H9c2 cells observed evenat its 10 �M concentration.

Dexrazoxane (ICRF-187, D-RZX) is used to protect againstcardiotoxicity that can develop as a serious side effect of anti-cancer chemotherapy with anthracyclines such as doxorubicin(adriamycin) or daunorubicin. The cardioprotective mechanism ofD-RZX has been traditionally attributed to intracellular Fe chela-tion by its EDTA-related hydrolysis product ADR925. In this study,ADR925 significantly suppressed ascorbate oxidation induced bycomplex of oxidized EPI + FAC in solution. However, in cellularexperiments, D-RZX has shown no potential to protect againstoxidative stress and toxicity of EPI, ISO or their oxidation products,even though it was tested over the wide concentration range of10–3000 �M. On the other hand, D-RZX has been previously shownto protect cardiomyocytes against hypoxia-reoxygenation injury(Hasinoff, 2002) and it significantly protected against the ISO car-diotoxicity in vivo (Flandina et al., 1990). Of note, lack of protectionby D-RZX against the ROS-mediated catecholamine-induced cellu-lar injury seen in the present study is in line with the recent notionthat D-RZX may protect against the anthracycline cardiotoxicityby mechanism(s) other than Fe chelation and antioxidant action(Popelova et al., 2009; Simunek et al., 2009). Like EDTA, ADR925is not selective for Fe and the observed protective effects in pre-vious studies may rather be attributable to calcium chelation andprevention of calcium overload of cardiac tissue (Simunek et al.,2005b).

Finally, two �-adrenoreceptor antagonist agents (propranololand carvedilol) and two dihydropyridine calcium channel block-ers (nifedipine and amlodipine) were assessed as these drugsare components of pharmacological cardioprotection followingmyocardial infarction, management of cardiac arrhythmias, con-gestive heart failure and/or hypertension. These agents block the�-adrenoreceptor and increase in Ca2+, respectively. Carvedilol andamlodipine are agents with anti-oxidant activity, whereas pro-pranolol and nifedipine do not possess considerable anti-oxidantactivity. Of note, both carvedilol and amlodipine have been previ-ously shown to protect cardiac cells against the cardiotoxicity ofanthracycline doxorubicin (Arozal et al., 2010; Spallarossa et al.,2004; Yamanaka et al., 2003). In our study though, none of these

agent has been shown to provide meaningful protection against thecatecholamine cardiotoxicity in our in vitro experimental setting.

In conclusion, small-molecular and lipophilic Fe chelatingagents L1 and ICL670A may represent an effective new treatment

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trategy for the protection of cardiac cells against catecholamine-nduced oxidative stress and toxicity even when Fe dysregulation isot the primary cause of myocardial injury. Further investigationsf L1 and ICL670A cardioprotective potential are warranted, prefer-bly using more complex in vivo models of cardiovascular diseasesith known or presumed role of catecholamine-mediated injury.

onflict of interest statement

The authors declare no competing financial interests.

cknowledgements

The authors thank Mrs. Alena Pakostová for her technicalssistance in the cell-culture laboratory and Dr. John ConnellyApoPharma, Inc.) for providing L1. This study was supported by theharles University in Prague (grants GAUK 51308/C/2008, GAUK67911/C/2011 and SVV 2011/263/004).

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