Doxorubicin-Dependent Reduction of Ferrylmyoglobin and Inhibition

Chem. Res. Toxicol. 2002, 15, 1179-1189

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Doxorubicin-Dependent Reduction of Ferrylmyoglobin and Inhibition of Lipid Peroxidation: Implications for Cardiotoxicity of Anticancer Anthracyclines Pierantonio Menna,†,‡ Emanuela Salvatorelli,†,‡ Rossella Giampietro,‡ Giovanni Liberi,§ Giovanni Teodori,§ Antonio M. Calafiore,§ and Giorgio Minotti*,‡ Departments of Drug Sciences and Cardiology and Cardiac Surgery, G. d’Annunzio University School of Medicine, Chieti, Italy Received June 21, 2002

Lipid peroxidation has been proposed to mediate cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines; however, there have been reports showing that DOX can also inhibit lipid peroxidation. Here we characterized the effects of DOX on the oxo-ferryl moiety [FeIVdO, MbIV] of H2O2-activated myoglobin, a lipid oxidant likely formed in the heart during treatment with DOX. MbIV was formed in vitro by reacting 100 µM H2O2 with 50 µM horse heart metmyoglobin (MbIII). Spectral studies showed that DOX reduced MbIV to MbIII, halfmaximal regeneration of MbIII occurring at ∼18 µM DOX. Comparisons between DOX, its aglycone doxorubicinone, and other approved or investigational anthracyclines or model compounds (daunorubicin, idarubicin, aclarubicin, and naphthazarin), showed that DOX reduced MbIV through the hydroquinone moiety of its tetracyclic ring. DOX inhibited MbIVdependent peroxidation of arachidonic acid, suppressing the formation of thiobarbituric acidreactive substances with an IC50 of ∼18 µM. Lipid peroxidation was inhibited also by the hydroquinone-containing daunorubicin and idarubicin but not by the hydroquinone-deficient aclarubicin; moreover, neither simple hydroquinone nor other known MbIV reductants (ascorbate, glutathione, and ergothioneine) reached measurable IC50s in a micromolar range. DOX-dependent inhibition of lipid peroxidation correlated with its ability to reduce MbIV to MbIII in competition with arachidonic acid (r ) 0.83, P ) 0.029); it did not correlate with its ability to scavenge other free radical species [like e.g., peroxyl radicals generated through the thermal decomposition of 2,2′-azo-bis(2-amidinopropane)]. DOX reduced MbIV and inhibited lipid peroxidation also when H2O2, MbIII and arachidonic acid were reacted in cytosol of human myocardial biopsies, a model developed to predict the cardiotoxic mode of action of DOX in patients. These results illustrate “antioxidant” properties of DOX, mediated by reduction of MbIV to MbIII, and cast doubts on lipid peroxidation as a causative mechanism of anthracyclineinduced cardiotoxicity.

Introduction Clinical use of doxorubicin (DOX)1 and other anticancer anthracyclines is limited by development of a lifethreatening cardiomyopathy upon chronic administration (1). One-electron redox cycling of a quinone moiety in DOX may concur in determining cardiotoxicity. In fact, one-electron reduction of the quinone, mediated by a variety of NAD(P)H oxidoreductases, yields a semiquinone which readily regenerates its parent compound * To whom correspondence should be addressed. Phone: 011-390871-3555320. Fax: 011-39-0871-3555356. E-mail: [email protected]. † P. M. and E. S. contributed equally to this paper. ‡ Department of Drug Sciences. § Department of Cardiology and Cardiac Surgery. 1 Abbreviations: DOX, doxorubicin; DNR, daunorubicin; IDA, idarubicin (4-demethoxydaunorubicin); aclarubicin, aclacynomycin A; naphthazarin, 5,8-dihydroxy-1,4-naphthoquinone; O2•-, superoxide anion; H2O2, hydrogen peroxide; •OH, hydroxyl radical; CAT0, catalase added at zero time; CAT3, catalase added after 3 min; MbIII, metmyoglobin; •Mb, myoglobin protein radical; MbIV, ferrylmyoglobin iron-oxo moiety; GSH, reduced glutathione; ergothioneine, 2-mercaptohistidine trimethylbetaine; TBA(RS), thiobarbituric acid (reactive substances); AAPH, 2,2′-azo-bis(2-amidinopropane) dihydrochloride; aminotriazole, 3-amino-1,2,4-triazole; IC50, 50% inhibitory concentration.

by reducing oxygen to O2•- and H2O2. At the same time, the semiquinone reductively releases iron from ferritin by direct or O2•--mediated electron transfer (2). These processes increase cellular levels of H2O2 and Fe(II) and set the stage for formation of •OH or iron-oxygen complexes known to initiate lipid peroxidation (3). The last years have witnessed growing interest in myoglobin (Mb) as an alternative source of iron for initiation of lipid peroxidation in diseased states characterized by increased formation of H2O2. In fact, H2O2 oxidizes the heme moiety of Mb to an oxoferryl species (FeIVdO), which is highly active in peroxidizing lipids (4). We will call this species MbIV. Hydrogen peroxideactivated Mb retains also a porphyrin π-cation radical which quickly dissipates in the globin in the form of delocalized tyrosine- or tryptophan-centered radicals (57). We will call this protein radical •Mb. Solid evidence for the participation of •Mb in the oxidation of a variety of organic substrates has been reported, but whether •Mb participates also in lipid peroxidation remains open to debate (4, 5). It is worth noting that MbIV formation occurs more favorably when H2O2 reacts with deoxyMbII

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or metmyoglobin (MbIII) rather than with MbIIO2 (8). Therefore, an ideal setting for formation of MbIV and initiation of lipid peroxidation has been identified in cardiac ischemia-reperfusion, a condition characterized by conversion of MbIIO2 to MbIII/deoxyMbII during ischemia and by formation of H2O2 during blood reflow (9, 10). Conditions favoring MbIV formation probably occur also when cardiomyocytes are exposed to anthracyclines. First, direct interactions of DOX with MbIIO2 accelerate oxygen dissociation and formation of MbIII, although the precise mechanisms of such interactions have remained undefined (11). Second, preferential accumulation of DOX in mitochondria (12), the most active site of anthracycline redox cycling (13), anticipates prolonged oxygen consumption and equilibration of some MbIIO2 with deoxyMbII. Finally, the concentration of Mb in cardiomyocytes (∼200 µM) (9) is several times higher than that of iron possibly released from ferritin (∼10-20 µM after several minutes of redox cycling in highly favorable in vitro systems) (2). All such factors highlight the ease with which MbIV might be formed in cardiac tissue exposed to DOX. Attempts to validate cause-effect relationships between lipid peroxidation and DOX-induced lipid peroxidation have produced positive (1) but also negative evidence. For example, some investigators have shown that laboratory animals may develop cardiomyopathy also in the absence of cardiac lipid peroxidation (reviewed in ref 14); others have shown that lipid soluble antioxidants prevent or delay cardiomyopathy in rodents but not in dogs or patients (reviewed in ref 15). We found that DOX infusions did not increase but actually suppressed cardiac lipid peroxidation in patients (16). While possibly explaining the lack of efficacy of lipid soluble antioxidants against the clinical pattern of DOX-induced cardiotoxicity, these studies offered unexpected evidence that DOX-popularly referred to as a prooxidant drugmight also blunt oxidative damage under defined conditions. Inhibition of cardiac lipid peroxidation was attributed to the fact that DOX generates H 2O2 in large excess over ferritin-released Fe(II). Under these conditions, the vast majority of H2O2 would remain available to interact with •OH and convert it into the less damaging O2•- (H2O2 + •OH f H2O + O2•- + H+) (3, 15). Alternatively, excess H2O2 would oxidize all ferritin-released Fe(II) to Fe(III), precluding formation of Fe(II):Fe(III) ratios which govern initiation of lipid peroxidation by iron-oxygen complexes with •OH-like reactivity (3, 15). Possible interactions of DOX with H2O2-activated Mb, inhibiting lipid peroxidation the same way as DOX inhibited lipid peroxidation mediated by ferritin-released Fe(II), have not been explored. Excess H2O2, generated through the redox cycling of the quinone moiety, would not be important in this setting, as lipid peroxidation actually increases with the molar ratio of H2O2 to MbIII (16). Excess H2O2 might degrade the heme pocket of Mb, but tissue damage then would occur through the redox intermediacy of iron released from the heme pocket (17). Reduction of MbIV to MbIII or MbIIO2 would be a more logical mechanism to preclude initiation of lipid peroxidation by H2O2-activated MbIII (18, 19). Therefore, we designed in vitro experiments to see whether DOX residues other than the quinone moiety had some type of reducing activity on MbIV, leading to inhibition of lipid peroxidation. This information would be of value to further appraise “antioxidant” properties of DOX and

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reevaluate the role of lipid peroxidation as a causative mechanism of anthracycline-induced cardiotoxicity.

Experimental Procedures Chemicals. DOX, daunorubicin (DNR), and idarubicin (IDA, 4-demethoxydaunorubicin) were obtained through the courtesy of Dr. Antonino Suarato (Department of Chemistry, PharmaciaUpjohn, Milan). Doxorubicinone was prepared by us after thermoacid hydrolysis of DOX, and purified on (2 × 10 cm) silicic acid columns equilibrated and eluted with CHCl3/CH3OH/CH3COOH (100:2:5). Purity was checked by 2D TLC on 0.25 mm (20 × 20 cm) Silica Gel F254 Plates (Merck, Darmstadt Germany), using CHCl3/CH3OH/CH3COOH (100:2:5) in 1D, and CH3COOC2H5-CH3CH2OH-CH3COOH-H2O (80:10:5:5) in 2D (Rf : 0.301D-0.722D) (20). Naphthazarin (5,8-dihydroxy-1,4naphthoquinone) and AAPH [2,2′-azo-bis(2-amidinopropane) dihydrochloride] were purchased from Fluka (Milan, Italy) and Polysciences Inc. (Warrington, PA), respectively. Thymol-free bovine liver catalase, aclacynomycin A (aclarubicin), horse heart MbIII, arachidonic acid (sodium salt), bovine serum albumin, and all other chemicals, were from Sigma-Aldrich (Milan, Italy). Spectral Studies. MbIV was formed in 1 mL incubations containing H2O2 and metmyoglobin (MbIII). Upon the basis of preliminary titrations within a broad range of H2O2:MbIII ratios, we chose to work with a 2:1 ratio (100 µM H2O2/50 µM MbIII), giving the highest and most stable yield of MbIV. MbIII was quantitated by assuming 630nm ) 3.5 mM-1 cm-1; MbIV was quantitated according to the formula: MbIV (mM) ) [(249 × A550nm) - (367 × A630nm)] (see ref 18 and references therein). MbIV reduction was calculated by monitoring absorbance decrease at 550 nm ( ) 3.1 mM-1 cm-1), and MbIII regeneration was monitored as absorbance increase at 630 nm (∆630nm ) 2.1 mM-1 cm-1) (18). Experiments were carried out at 37 °C in 0.3 M NaCl, carefully adjusted to pH 7.0, to avoid interferences of most common buffers with anthracycline reactions (21, 22). Although unbuffered, the pH of incubations did not vary throughout the experiment. These standard conditions were modified in experiments with doxorubicinone, the aglycone of DOX, which was vacuum-dried and dissolved in ethanol due to its poor solubility in water. These experiments were performed by keeping in mind that ethanol is a reducing substrate for MbIV (23); therefore, ethanol never exceeded 5-6 µL/mL, an amount which had no effect on the decay of MbIV to MbIII (not shown). Similar precautions were taken in experiments with naphthazarin, dissolved in dimethyl sulfoxide. All studies were performed in a Hewlett-Packard 8453A diode array spectrophotometer, with computer-assisted corrections for background absorbance of anthracyclines in the 400-600 nm range. Human Cardiac Cytosol. Small myocardial samples (∼0.1 g) were taken from the lateral aspect of excluded right atrium of patients undergoing aorto-coronary bypass grafting. All specimens were routinely disposed of by the surgeons during cannulation procedures; therefore, patients were not subjected to any unjustified or ethically unacceptable loss of tissue (24). After storage at -80 °C, pools of 10-15 anonymous biopsies were processed for cytosol preparation by sequential homogenization, 20-min centrifugations at 16000 and 25000g, and 90min ultracentrifugation at 105000g. Next, 105000g supernatants were subjected to overnight stirring with 65% ammonium sulfate, followed by 20-min centrifugation at 16000g to precipitate proteins. This procedure removed endogenous myoglobin by “salting out” (10), but improved the stability of cytosol upon freezing-thawing and prevented development of excess turbidity when cytosol was reconstituted with DOX for spectral studies (25). Cytosolic proteins were suspended in a minimum volume of 0.3 M NaCl, and subjected to dialysis against two 1-L changes of 0.3 M NaCl-1 mM EDTA to remove adventitious iron and other low molecular weight contaminants. EDTA-iron complexes were eventually removed by dialysis against two 1-L changes of 0.3 M NaCl (25). Lipid Peroxidation. Lipid peroxidation was studied in incubations (0.25-0.5 mL final volume) containing 50 µM MbIII,

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Figure 1. Effects of DOX on MbIV. (A) Time-dependent oxidation of MbIII to MbIV by H2O2. (B) Spontaneous decay of MbIV in the absence or presence of CAT3. (C) H2O2-dependent spectral changes of MbIII in the presence of DOX. (D) Spontaneous decay or DOXinduced reduction of MbIV, in the absence or presence of CAT3 (2600 units/mL). DOX and MbIII were 50 µM; H2O2 was 100 µM. Other conditions were as described in the Experimental Procedures. 0.4 mM arachidonic acid (dissolved in ethanol as described for doxorubicinone), in the absence or presence of increasing concentrations of anthracyclines or other test compounds, in 0.3 M NaCl, pH 7.0. Human cardiac cytosol (1 mg prot./mL) was also included in selected experiments. Reactions were started by adding H2O2 (100 µM). Where indicated, lipid peroxidation was induced by replacing MbIII and H2O2 with the free radical initiator AAPH. These latter experiments had to be performed in phosphate-buffered saline to ensure adequate buffering of AAPH (5 mM). After 30 min of incubation at 37 °C, the reaction mixtures were assayed for thiobarbituric acid-reactive substances (TBARS) against appropriate blanks, with modifications involving butanol extraction of the TBA adduct (26). The antioxidant butylated hydroxytoluene (0.03 vol in 2% ethanol) was included in the TBA reagent to prevent further peroxidation of lipids during the assay (26). Control experiments, performed by adding DOX to the TBA reagent at the end of 20 min incubations of arachidonic acid with H2O2/MbIII or AAPH, showed that the anhracycline did not interfere with with the assay. Other Assays and Analyses. Proteins were assayed by the bicinchoninic acid method (27), and catalase was assayed according to Beers and Sizer (28). Unless otherwise indicated, values are given as means ( SE. Differences between two sets of data were analyzed by one-tailed unpaired Student’s t-test; differences between >2 sets of data were analyzed by one-way ANOVA followed by Bonferroni’s test for multiple comparisons. Correlations were determined by one-tailed nonparametric Spearman analysis. Differences and correlations were considered to be significant when P < 0.05.

Results DOX Reduces MbIV. Figure 1A shows the spectrum of 50 µM MbIII, characterized by distinct peaks at 502, 582, and 630 nm. The addition of 100 µM H2O2 caused time-dependent disappearance of these peaks and appearance of new absorption maxima at 546 and 586 nm, indicative of oxidation of MbIII to MbIV (18). MbIV formation reached its maximum at 3 min; at this time point MbIV averaged 51 ( 0.1 µM (n ) 10), calculated according to the algorithm reported under Experimental Procedures, indicating a complete oxidation of MbIII by H2O2. Time-course experiments at 550 nm (Figure 1B) also

showed that MbIV remained stable for at least 15-20 more minutes; this demonstrated that at the 2:1 ratio of H2O2 to MbIII used in our study there was no secondary reduction of MbIV by residual H2O2, a process which we might have seen if MbIV acted as a catalase mimetic (29). In contrast, we found that residual H2O2 acted as an oxidant which prevented a spontaneous decay of MbIV to MbIII. This was evidenced by the fact that adding catalase 3 min after reaction of MbIII with H2O2sa condition hereafter referred to as CAT3scaused a moderate but progressive decrease of absorbance at 550 nm (Figure 1B). Spectra recorded 20 min after mixing MbIII with H2O2, in the absence or presence of CAT3, are shown in Figure 1C. In accordance with the time courses at 550 nm, CAT3 induced a moderate decay of the spectrum of MbIV. This pattern changed when MbIII and H2O2 were reacted in the presence of 50 µM DOX. As also shown in Figure 1C, DOX caused the appearance of a spectrum which reflected a composite of MbIII and MbIV, evidenced by multiple peaks at 502, 546, and 586 nm; however, the addition of CAT3 enabled DOX to regenerate the same spectrum of unreacted MbIII. Time-course experiments at 550 nm showed that DOX did not appreciably interfere with MbIV formation; instead, DOX enhanced MbIV decay, both in the absence and especially in the presence of CAT3 (Figure 1D). Under the latter condition, MbIV reduction and MbIII regeneration were enhanced by DOX in a concentration-dependent manner. The effects of DOX on the initial rates of MbIV reduction and MbIII regeneration, calculated after addition of CAT3, were linear up to 50 µM, half-maximal stimulation occurring at ∼30 µM. Total recovery of MbIII after 20 min incubation also increased linearly with the concentration of DOX, halfmaximal recovery occurring when DOX was ∼18 µM (Figure 2). These results demonstrated that DOX reduced MbIV to MbIII, a process which became more evident when CAT3 was used to decompose residual H2O2 and to prevent reoxidation of MbIII to MbIV. Structure-Activity Studies. We performed experiments for identifying MbIV reductant(s) in the anthracycline molecule. As shown in Chart 1, DOX is composed

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Figure 2. DOX-induced concentration-dependent reduction of MbIV and regeneration of MbIII in the presence of CAT3. MbIV was formed as in Figure 1, in the presence of increasing concentrations of DOX. Initial rates of MbIV reduction were determined after addition of CAT3 (2600 units/mL), as described in the Experimental Procedures. Values are taken from representative experiments.

of aglyconic and sugar moieties. The aglycone (doxorubicinone) is composed of a tetracyclic ring with adjacent quinone-hydroquinone moieties in rings C-B, a methoxy group at C-4 in ring D, and a short side chain which originates at C-8 in ring A and contains a carbonyl group at C-13 and a primary alcohol at C-14. The sugar (daunosamine) is attached by a glycosidic bond to C-7 in ring A and consists of a 3-amino-2,3,6-trideoxy-L-fucosyl moiety. As also shown in Chart 1, 50 µM doxorubicinone was even more effective (P < 0.01) than equimolar DOX in reducing MbIV. While indicating that daunosamine probably limited sterical approach of the anthracycline to the hemeprotein, these results clearly implied that MbIV reductant(s) were not located in the aminosugar moiety. We searched for other reducing site(s) in the anthracycline molecule, keeping in mind the following reports in the literature: (i) the side-chain primary alcohol is able to reduce nonheme Fe(III) coordinated in 3:1 anthracycline:Fe(III) complexes (30); (ii) hydroquinone compounds are good reductants for MbIV (18, 19); (iii) Compounds I and II of horseradish peroxidase, sharing similarities with H2O2-activated Mb, can catalyze Odemethylation of a variety of xenobiotics (31). We therefore focused on the MbIV-reducing properties of the primary alcohol and methoxy and hydroquinone residues of DOX. This was done by comparing 50 µM DOX to equimolar analogues such as DNR, in which the sidechain primary alcohol is replaced by a methyl; its 4-demethoxy derivative IDA; and aclarubicin, an anthracycline having the ring B hydroquinone replaced by a phenolate (cf. Chart 1). Both DNR and IDA reduced MbIV as effectively as DOX, demonstrating that neither the primary alcohol nor the methoxy group were involved in donating electrons to MbIV. In contrast, the hydroquinone-deficient aclarubicin was remarkably less effective (P < 0.001) than DOX or other anthracyclines in reducing MbIV, even when its concentration was raised to 100 or 200 µM. The limited activity of aclarubicin was only in part explained by sterical interferences caused by the presence of a trisaccharide moiety; in fact, aclarubicin was less effective than DOX even after prolonged thermoacid hydrolysis, a procedure which converted it into a mixture of aglyconic/monosaccharide derivatives [MbIV reduction (µM/min) at 50 µM anthracyclines: 1.9 (hydrolyzed aclarubicin) vs 5.3 (DOX)]. These results

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showed that the hydroquinone moiety accounted for most of the MbIV-reducing activity of DOX. Accordingly, excellent rates of MbIV reduction-virtually comparable to those induced by doxorubicinone- were obtained with naphthazarin, a model compound reproducing the quinonehydroquinone central portion of doxorubicinone (Chart 1). Comparisons between naphthazarin and doxorubicinone also revealed that neither ring D nor ring A or its side chain introduced significant sterical restrictions in hydroquinone/MbIV interactions. MbIV-Dependent Lipid Peroxidation. Arachidonic acid (0.4 mM) accelerated the decay of MbIV generated in MbIII/H2O2/CAT3 incubations, as evidenced by timedependent decrease of peaks at 546 and 586 nm (Figure 3, panels A and B). Direct comparisons of spectra recorded 30 min after mixing MbIII and H2O2 showed that arachidonic accelerated regeneration of MbIII from MbIV, as the decrease in absorbance at 546 and 586 nm was accompanied by recovery of MbIII absorbances at 502 and 630 nm (Figure 3C). Once corrected for the spontaneous decay of MbIV, the initial rates of MbIV reduction induced by 0.4 mM arachidonic acid were much lower than those induced by as little as 50 µM DOX (1.1 ( 0.1 vs 5.2 ( 0.1 µM/min; n ) 9 or 12, respectively, P < 0.001). Nonetheless, these findings offered evidence for hydrogen abstraction and electron transfer from the lipid to MbIV. This process would generate lipid alkyl radicals which initiate lipid peroxidation by adding molecular oxygen (3); therefore, we measured TBARS in incubations containing arachidonic acid plus H2O2 and/or MbIII. As shown in Figure 3D, neither H2O2 nor MbIII converted arachidonic acid to TBARS. This demonstrated that H2O2 and MbIII lacked sufficient reactivity to abstract hydrogen from arachidonic acid and also showed that arachidonic acid did not contain preformed lipid hydroperoxides which might have been decomposed by MbIII to lipid alkoxyl radicals (3). Lipid peroxidation occurred when arachidonic acid was incubated with both H2O2 and MbIII, a finding consistent with initiation by MbIV. Accordingly, lipid peroxidation was suppressed by adding catalase at zero time (CAT0), a condition favoring rapid decomposition of H2O2 before it converted MbIII to MbIV, but not by adding CAT3, which only caused moderate decay of MbIV formed by prior reaction of MbIII with H2O2 (see also Figure 3D and cf. Figure 1, panels B and C). Moreover, the iron chelator EDTA had no effect on TBARS formation in the H2O2/MbIII/CAT3 system, demonstrating that lipid peroxidation did not proceed after heme degradation and consequent release of iron ions. DOX Inhibits MbIV-Dependent Lipid Peroxidation. We determined the effects of DOX on lipid peroxidation induced by MbIV in the H2O2/MbIII/CAT3 system. As shown in Figure 4A, DOX inhibited MbIV-dependent lipid peroxidation in a concentration-dependent manner, with an IC50 of ∼18 µM; this value reproduced the concentration of DOX inducing ∼50% regeneration of MbIII from MbIV (cf. Figure 2). Lipid peroxidation was similarly inhibited by hydroquinone-containing DNR and IDA, but not by the hydroquinone-deficient analogue aclarubicin, not even when its concentration was raised to 200 µM (see also Figure 4A). These results suggested that DOX inhibited lipid peroxidation by reducing MbIV through the hydroquinone moiety in its tetracyclic ring. However, neither simple hydroquinone nor other established MbIV reductantsslike ascorbate, GSH, and the naturally occurring thiolhistidine ergothioneine (9)s



Chart 1. Anthracycline-Dependent MbIV Reduction: Comparisons between DOX and Analoguesa

a All anthracyclines were tested at 50 µM; aclarubicin was tested also at 100 and 200 µM. The arrows indicate hydrolytic removal of daunosamine in doxorubicinone; replacement of the side-chain primary alcohol with a methyl in DNR; lack of both primary alcohol and 4-demethoxy residues in IDA; lack of the hydroquinone in aclarubicin. Values are given as initial rates, corrected for the spontaneous decay of MbIV, and are means ( SE of 3-12 experiments (except values for 100 and 200 µM aclarubicin or 50 µM naphthazarin, which were taken from representative experiments).

reached a measurable IC50 when assessed in a 5-100 µM range (Figure 4B). We considered that the greater potency of DOX at inhibiting lipid peroxidation reflected some type of free radical-scavenging activity not directly related to MbIV reduction. Therefore, we determined whether DOX inhibited lipid peroxidation induced by peroxyl radicals generated through the thermal decomposition of AAPH (32). Direct comparisons with MbIVdependent peroxidation were obtained by adjusting AAPH to 5 mM, a concentration at which peroxyl radicalinduced peroxidation yielded approximately ∼90% of

TBARS usually produced by the H2O2/MbIII/CAT3 system (3.3 ( 0.5 µM vs 3.7 ( 0.1 µM; n ) 6). As shown in Figure 5A, not only DOX but also simple hydroquinone and other electron donors inhibited AAPH-dependent peroxidation of arachidonic acid when assessed at 5-100 µM, exhibiting IC50s within a relatively narrow range (DOX ∼9 µM; simple hydroquinone and GSH, ∼10 µM; ergothioneine, ∼30 µM; ascorbate, ∼35 µM). Having obtained this information, we determined whether the inhibitory effects of DOX and other reductants on MbIV-induced peroxidation correlated with their free radical-trapping

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Figure 3. Reactions of MbIV with arachidonic acid. (A) MbIV was formed as in Figure 1, and spectra were recorded immediately after addition of CAT3 (2600 units/mL) and every 90 s up to 30 min. (B) Same conditions as in panel A, except that incubations contained also arachidonic acid (0.4 mM). (C) Comparisons of spectra recorded at 30 min in incubations with or without arachidonic acid. (D) TBARS were measured after 20 min incubation of arachidonic acid with H2O2 and/or MbIII, precisely as described in the Experimental Procedures. Where indicated, incubations also contained CAT0 or CAT3 (both 2600 units/mL), or EDTA (200 µM) plus CAT3. Values are means ( SE of three experiments; those without verticals bars have SE within columns.

Figure 4. Effects of anthracyclines and other reducing substrates on arachidonic acid peroxidation induced by H2O2/MbIII/ CAT3. Incubations were prepared as in Figure 3D. Where indicated, they also contained increasing concentrations of DOX, DNR, IDA, and aclarubicin (panel A), or other reducing compounds (panel B). Values are means ( SE of three experiments; those without verticals bars have SE within symbols.

activity, expressed as inhibition of AAPH-induced lipid peroxidation. Figure 5B illustrates data obtained at 50 µM test compounds and shows that the free radicaltrapping activity of DOX and other reductants did not correlate with their efficacy at inhibiting lipid peroxidation induced by MbIV. We therefore considered that the unique ability of DOX to inhibit MbIV-dependent lipid peroxidation reflected its unusually higher efficacy at reducing MbIV to MbIII as compared to other electron donors. This proved to be the

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Figure 5. Inhibition of MbIV-dependent lipid peroxidation by DOX and other reducing substrates: correlations with free radical trapping activity. (A) Inhibition of AAPH-dependent arachidonic acid peroxidation by DOX or other reducing compounds (values taken from duplicate experiments with >95% agreement). (B) Correlation between free radical trapping activity (expressed as percent inhibition of AAPH-dependent lipid peroxidation) and inhibition of MbIV-dependent lipid peroxidation (calculated from mean values in Figure 4). All data were calculated at 50 µM test compounds. See also text for further explanations.

case; in fact, comparative experiments at 50 µM showed that DOX was remarkably more effective (P < 0.01) than simple hydroquinone and other electron donors in reducing MbIV to MbIII [net reduction rates (µM/min): ascorbate, 0.8 ( 0.3; hydroquinone, 1.8 ( 0.1; GSH, 2.8 ( 0.4; ergothioneine, 3.4 ( 0.3; DOX, 5.2 ( 0.1; n ) 3-5]. Importantly, 50 µM DOX was shown to promote a complete conversion of MbIV to MbIII also in the presence of 0.4 mM arachidonic acid, demonstrating that it was able to compete with the lipid as an electron donor for the oxoferryl moiety; under comparable conditions, hydroquinone and other reductants failed to promote a complete reduction of MbIV to MbIII (Figure 6A). Calculations of the initial rates of MbIV reduction, in the presence of arachidonic acid, showed that DOX was more effective than other reducing agents at all concentrations tested in this study (Figure 6B). Moreover, the greater efficacy of DOX at inhibiting MbIV-dependent lipid peroxidation correlated in a significant manner with its higher ability to reduce MbIV in competition with arachidonic acid (Figure 6C). Studies in Human Cardiac Cytosol. MbIII (50 µM) and H2O2 (100 µM) were reacted in human cardiac cytosol to see whether DOX reduced MbIV and inhibited lipid peroxidation under conditions reproducing a tissue environment. These experiments were not confounded by the presence of endogenous myoglobin, which had been removed by “salting out” after 65% ammonium sulfate precipitation of other cytosolic proteins (cf. Experimental Procedures). As shown in Figure 7A, cytosolic proteins reduced the maximum yield of MbIV and accelerated its decay with time. These effects were concentration-dependent, approaching saturation at ∼1-3 mg of protein/ mL, and seemed to be quite specific to cytosolic protein(s),



Figure 6. Inhibition of MbIV-dependent lipid peroxidation by DOX and other reducing compounds: correlations with MbIV reduction. (A) MbIV was formed in the H2O2/MbIII/CAT3 system and spectra were taken at 20 min; where indicated, incubations also contained arachidonic acid (0.4 mM), or arachidonic acid plus 50 µM DOX or other test compounds. (B) Initial rates of MbIV reduction in the H2O2/MbIII/CAT3 system. All incubations contained arachidonic acid (0.4 mM) and increasing concentrations of DOX or other test compounds. Values are means ( SE of three to four experiments; those without vertical bars have SE within symbols. (C) Correlation between inhibition of MbIV-dependent lipid peroxidation and net stimulation of MbIV reduction by DOX or other test compounds in the presence of 0.4 mM arachidonic acid. Data were calculated at 50 µM test compounds, and were taken from mean values in panel B and Figure 4. See also text for further explanations.

Figure 7. Reactions of MbIII with H2O2 in human cardiac cytosol. (A, B) Formation of MbIV in the absence or presence of increasing amounts of cytosol or bovine serum albumin. (C) Time course of MbIV formation and decay in the presence of 3 mg of protein of cytosol/mL, with or without aminotriazole. (D) Spectra recorded 2 and 20 min under the same conditions of panel C; to permit better visualization of low level MbIV, spectra at 2 min were obtained by correcting MbIII-MbIV composites for background absorbance of unreacted MbIII. MbIII and H2O2 were 50 and 100 µM, respectively; aminotriazole was 1 mM.

as replacing cytosol with equal amounts of albumin had much less evident effects on the yield and decay of MbIV (Figure 7B). These experiments were performed in the absence of CAT3; therefore, we assessed whether the reduced detection and accelerated decay of MbIV reflected decomposition of H2O2 by CAT present in the cytosol (177 units/mg of protein). This information was obtained by

replicating experiments in the presence of 1 mM aminotriazole, a widely used catalase inhibitor, which blunted enzyme activity to 9.3 units/mg of protein. Aminotriazole did improve MbIV detection when MbIII and H2O2 were reacted in the presence of 3 mg of protein of cytosol/mL (see time course and spectra at 2 min, Figure 7, panels C and D). Nonetheless, the maximum yield of MbIV

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Figure 8. DOX-induced MbIV reduction and inhibition of lipid peroxidation in human cardiac cytosol. (A) MbIV was formed by reacting 50 µM MbIII and 100 µM H2O2 in the presence or absence of 1 mg of protein of cytosol/mL. All incubations also contained 1 mM aminotriazole. Where indicated, 50 µM DOX and/or 0.4 mM arachidonic acid were added, and MbIV reduction was calculated over the linear phase of the reaction (usually 90-180 s). (B) TBARS were measured in human cardiac cytosol (1 mg of protein/mL) supplemented with 0.4 mM arachidonic acid and exposed to 50 µM MbIII and/or 100 µM H2O2. (C) The complete system (cytosol, arachidonic acid, and MbIV) was assayed for TBARS in the presence of increasing concentrations of DOX. Values are means of two separate determinations with >80% agreement.

remained ∼35% of that observed in the absence of cytosol, nor could aminotriazole prevent the accelerated decay of MbIV to MbIII (see also time course and spectra at 20 min, Figure 7, panels C and D). Reactions of H2O2 with other cytosolic (iron)protein(s), and/or redox coupling of MbIV with electron-rich residues of target protein(s), probably contributed to a greater extent in reducing detection and stability of MbIV in the cytosol. Having seen these effects, we determined whether DOX retained an ability to reduce MbIV also in the cytosol. This was done by reconstituting DOX, MbIII, and H2O2 with 1 mg of protein of cytosol/mL; aminotriazole (1 mM) was also included to both optimize MbIV detection and simulate conditions when DOX generates H2O2 beyond the detoxifying activity of catalase (15). As shown in Figure 8A, 50 µM DOX strongly enhanced the initial rates of MbIV reduction in the cytosol; some enhancement was observed also with 0.4 mM arachidonic acid, but net stimulation of MbIV reduction by arachidonic did not exceed 10% of that afforded by DOX. This latter estimate was in good agreement with earlier determinations in cytosol-free incubations, according to which 0.4 mM arachidonic acid was ∼80% less effective than 50 µM DOX in reducing MbIV. Moreover, combining arachidonic with DOX gave essentially the same rate of MbIV reduction as that mediated by DOX per se, demonstrating that DOX was able to compete with both protein and lipid substrates for MbIV. These results anticipated that (i) arachidonic acid was liable to peroxidation by MbIV generated in human cardiac cytosol, and (ii) DOX was able to inhibit such peroxidation by competing for MbIV and reducing it to MbIII. Figure 8B confirms that 50 µM MbIII plus 100 µM H2O2, but not MbIII or H2O2 added individually, converted arachidonic acid to TBARS in human cardiac cytosol, consistent with initiation of lipid

peroxidation by MbIV but not by other protein(s) of the cytosol. Interestingly, the yield of TBARS was ∼50% of that usually observed in cytosol-free incubations, although the presence of cytosol reduced the maximum yield of MbIV by ∼70%. (cf. Figures 4, 7, and 8). Control experiments, conducted in cytosol-free incubations and performed by titrating MbIII with a broad range of H2O2 concentrations, confirmed that lipid peroxidation reached its half-maximal level when approximately ∼30% of MbIII oxidized to MbIV (not shown). Lipid peroxidation, induced by MbIV in the presence of cytosol, was inhibited by DOX in a concentration-dependent manner, with an IC50 of ∼20 µM (Figure 8C).

Discussion We have shown that DOX reduces MbIV to MbIII. Complete reduction of MbIV to MbIIO2 was not observed during the course of this study, not even when DOX was incubated directly with MbIII; only ascorbate and hydroquinone proved to reduce MbIV to MbIIO2 but this occurred in a millimolar range (not shown). Nonetheless, limited reduction of MbIV to MbIII enabled DOX to prevent lipid peroxidation which otherwise occurred after spectrally detectable hydrogen abstraction and electron transfer from arachidonic to MbIV (cf. Figures 3 and 4). Structure-activity studies indicate that a hydroquinone moiety in the tetracyclic ring is important in mediating MbIV reduction by DOX; therefore, MbIV-dependent lipid peroxidation is inhibited also by DNR and IDA, two hydroquinone-containing analogues, but not by aclarubicin, a hydroquinone-deficient analogue. This latter observation introduces one more argument against the role of lipid peroxidation in anthracycline-induced cardiotoxicity, as aclarubicin proved to be more cardiac


tolerable than DOX and other anthracyclines in preclinical and early clinical evaluation (33). Comparative studies also show that DOX is remarkably more effective than simple hydroquinone at inhibiting lipid peroxidation induced by MbIV, and the same holds true when DOX is compared to other established MbIV reductants such as ascorbate, GSH, or ergothioneine. Our data show that the greater efficacy of DOX against MbIV-dependent lipid peroxidation does not correlate with nonspecific free radical-scavenging properties (cf. Figure 5); it only correlates with its higher activity in competing with arachidonic acid for MbIV, regenerating MbIII much faster than do simple hydroquinone or other potential electron donors (cf. Figure 6). How DOX engages in redox reactions with MbIV remains to be established. Data on the redox potential of the hydroquinone moiety in DOX or related anthracyclines are not available, and reported pKa values for the first ionization of this moiety (from 8 to 9.6) would not anticipate facile oxidation at physiologic pH (34). Some investigators have suggested that the pKa may decrease after interactions between the amino group in daunosamine and the -OH at C-6 in ring B (35), but this picture has been questioned by others (36). In our study doxorubicinone was more effective than DOX in reducing MbIV to MbIII, as if daunosamine introduced sterical restriction rather than redox facilitation in anthracyclineMbIV interactions (cf. Chart 1). A similar pattern of enhanced MbIV reduction was observed by replacing DOX with naphthazarin, a model compound reproducing the central quinone-hydroquinone portion of doxorubicinone, but this finding might have been contributed also by a reportedly lower pKa for naphthazarin vs other anthracyclines (34, 37). Uncertainties or lack of information on the redox characteristics of the hydroquinone moiety of anthracyclines preclude further speculation in this setting. At this time it is worth noting that half-maximal or near-to-complete regeneration of MbIII from ∼50 µM MbIV occurred when DOX was ∼18 or ∼38 µM, respectively (cf. Figure 2), as if one DOX always reduced more than one MbIV. On the basis of pulse radiolysis studies of semioxidized forms of naphthazarin (37), one can envision a sort of chain reaction in which DOX reduces MbIV, yielding MbIII and a hydroquinone-derived semiquinone which quickly disproportionates with another semiquinone to generate a ring C-B diquinone and regenerate a hydroquinone (reactions 1 and 2). Thus, redox coupling of DOX with MbIV is accompanied by 50% recycling of the anthracycline in its active hydroquinone form, possibly explaining how the stoichiometry of MbIV reduction vs DOX oxidation exceeded unity.

DOX-hydroquinone + MbIV f DOX-semiquinone + MbIII (1) DOX-semiquinone + DOX-semiquinone f DOX-diquinone + DOX-hydroquinone (2) Attempts to validate reactions 1 and 2, while also refining related stoichiometries, were made difficult by the fact that MbIII regeneration was accompanied by rapid (∼5 min) and complete loss of the optical, fluorescent, and HPLC/TLC chromatographic properties of DOX, a finding consistent with irreversible modification(s) of both parent anthracycline and diquinone derivative(s). This process was observed also with DNR and IDA or simple naph-


thazarin, suggesting that anthracyclines underwent chromophore degradation at rings C-B; a less extensive degradation (∼40% at 200 µM) was observed with the hydroquinone-deficient aclarubicin having limited reactivity toward MbIV (not shown). We have preliminary data to rule out an involvement of •Mb in anthracycline degradation. In fact, acetylation of MbIII over its tyrosine residues abolished H2O2-dependent oxidation of peroxidatic substrates [like 2,2′-diazinobis(3-ethylbenzothiazoline-6-sulfonic acid)], but did not prevent DOX degradation; moreover, there was no degradation of the anthracycline molecule when DOX was exposed to a sustained flux of tryptophan-derived radicals, generated by oxidizing millimolar amounts of tryptophan with H2O2/horseradish peroxidase (not shown). DOX degradation therefore seems to occur during coupled hydroquinone oxidation/ MbIV reduction. We are exploring whether diquinones undergo ring opening and fragmentation (34); as diquinones are strong electrophiles, we are also exploring whether chain reactions involve and oxidize DOX recycled from semiquinone disproportionation. This would explain how neither DOX nor diquinones could be recovered from H2O2/MbIII/DOX incubations. Electron spray ionization/mass spectroscopy studies have not allowed us to unambiguously identify anthracycline oxidation and/or fragmentation products; similar problems have been encountered by Reszka et al. (34) in their studies of DOX degradation by •NO2 radical generated through H2O2/lactoperoxidase/NO2-. MbIV has been detected by reflectance spectroscopy in rat isolated heart subjected to ischemia-reperfusion (9). This technique requires perfusion with millimolar Na2S to regenerate MbII and derivatize the porphyrin ring as SMbII (9). Similar procedures could not be used to visualize consecutive formation and reduction of MbIV by DOX, as Na2S might have reacted also with the labile anthracycline molecule and altered its redox behavior in the system. Moreover, we considered that the action of DOX is confined to reducing MbIV to MbIII, whose subsequent reaction with Na2S would be too slow to form SMbII within the experiment time (9). To obtain information of preclinical value, we demonstrated that DOX reduced MbIV and inhibited lipid peroxidation also when known amounts of H2O2 and MbIII were reacted in cytosolic fractions of human myocardial samples. In the past, we have used this model to predict cardiac formation of C-13 secondary alcohol metabolites by cytoplasmic aldo/keto- or carbonyl- reductases during the course of anthracycline regimens (20, 24, 25, 38). Here, the same model proved useful to show that cytosolic protein(s) can both compete with MbIII for H2O2 and reduce MbIV to MbIII, thereby decreasing the yield of MbIV and accelerating its decay. Nevertheless, DOX has sufficient MbIVreactivity to compete with cytosol and further accelerate MbIV reduction, inhibiting lipid peroxidation with an IC50 which compares to that determined in the absence of cytosol (∼20 vs ∼18 µM, respectively). In conclusion, we have shown that DOX, popularly referred to as a pro-oxidant drug, may act also as an “antioxidant” which inhibits lipid peroxidation. Whereas the prooxidant behavior of DOX is attributed to oneelectron reduction of the quinone moiety, its antioxidant behavior seems to originate from oxidation of the hydroquinone coupled with reduction of a lipid-damaging species such as MbIV. In light of the abundance of Mb in cardiomyocytes and of its possible role as a catalyst of

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lipid peroxidation, these results identify a novel mechanism to further explain inhibition of cardiac lipid peroxidation in cancer patients treated with DOX (16) and the consequent lack of clinical protection by lipidsoluble antioxidants such as vitamin E (15, 39). Possible toxicity of DOX oxidation and degradation product(s) toward cellular constituents other than lipids [e.g., enzymes with labile -SH groups (40)], cannot be ruled out at this time. In reconciling our previous and present data with the “lipid peroxidation hypothesis” of anthracycline-induced cardiotoxicity, we would note that induction of lipid peroxidation in isolated heart or cardiomyocytes usually requires concentrations of DOX several times higher than the plasma peak measured in patients after i.v. doses of the anthracycline; lipid peroxidation does not occur when the concentration of DOX used in vitro reproduces plasma levels observed in vivo (14, 41). Here we have shown that lipid peroxidation induced by H2O2-activated 50 µM MbIIIsa concentration well in the range of those occurring in cardiac tissuesis inhibited by DOX with IC50s of ∼1820 µM, in reconstituted systems as well as in human cardiac cytosol. These values compare with the concentrations of DOX measured in the heart of rodents 1 or 4 h after i.v. doses of the anthracycline that reproduce clinical regimens [∼22 or 13 nmol/g, respectively, corresponding to ∼22 or 13 µM if one considers that cardiac tissue has a density very similar to that of water (1 g/mL)] (42). Therefore, the “antioxidant” behavior of DOX is seen at concentrations achievable under pharmacokinetic conditions. The oxidative hypothesis of anthracycline-induced cardiotoxicity clearly remains of central interest to explain other modes of action of these drugs in the heart. The observation that cardiomyocytes are ill-equipped with oxyradical-detoxifying enzymesslike superoxide dismutase, catalase, or glutathione peroxidaseshelps to explain how DOX becomes toxic to the heart but not to other tissues (1, 14, 15). Likewise, the protective efficacy of cardiac-restricted catalase overexpression in transgenic mice (43), and the therapeutic benefit of treating laboratory animals or patients with the cell permeable iron chelator dexrazoxane (15, 44), lend unequivocal support to the involvement of reactive oxygen species and iron in inducing cardiomyopathy. Results described here and in preceding reports suggest that the precise nature of such oxidative damage should not be searched in nor confined to an upset of lipid peroxidation. In this respect, we have recently shown that iron and reactive oxygen species, possibly in concert with the secondary alcohol metabolite doxorubicinol, oxidatively damage and inactivate iron regulatory proteins which modulate the fate of mRNA for transferrin receptor or ferritin (45). Consequent alterations of cellular iron uptake vs sequestration may be followed by lipid-peroxidation-independent processes such as iron misplacement and sterical occupation of calcium channels in the sarcoplasmic reticulum, impairing contractility (46). Severe dysfunction of iron regulatory protein activities may be followed also by anomalous compensatory mechanismsslike uncontrolled derepression of ferritin synthesisswhich make cell develop apoptosis through global impairment of irondependent metabolism (47). These are few examples of how iron and reactive oxygen species might cause cardiotoxicity after attack to cellular constituents other than lipids, reconciling protection by iron chelators or oxyradi-

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cal-detoxifying measures with the lack of protection by lipid soluble antioxidants.

Acknowledgment. This work was supported by AIRC, MURST COFIN 2001, and MURST “Center of Excellence on Aging at the University of Chieti” (to G.M.).

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