Oxidative degradation of cardiotoxic anticancer anthracyclines to phthalic acids. Novel function or ferrylmyoglobin.

We show that the pseudoperoxidase activity of ferrylmyoglobin (MbIV) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. MbIV, formed in vitro by reacting horse heart MbIII with H2O2, caused disappearance of the spectrum of DOX at 477 nm and appearance of UV-absorbing chromophores that indicated opening and degradation of its tetracyclic ring. Electron spray ionization mass spectrometry analyses of DOX/MbIV ultrafiltrates showed that DOX degradation resulted in formation of 3-methoxyphthalic acid, the product of oxidative modifications of its methoxy-substituted ring D. Other methoxy-substituted anthracyclines similarly released 3-methoxyphthalic acid after oxidation by MbIV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and MbIII/H2O2 or hemin/H2O2 showed that the porphyrin radical of MbIV-compound I and the iron-oxo moiety of MbIV-compound II were sequentially involved in oxidizing DOX; however, oxidation by compound I formed more 3-methoxyphthalic acid than oxidation by compound II. Sizeable amounts of 3-methoxyphthalic acid were formed in the heart of mice treated with DOX, in human myocardial biopsies exposed to DOX in vitro, and in human cardiac cytosol that oxidized DOX after activation of its endogenous myoglobin by H2O2. Importantly, H9c2 cardiomyocytes were damaged by low concentrations of DOX but could tolerate concentrations of 3-methoxyphthalic acid higher than those measured in murine or human myocardium. These results unravel a novel function for MbIV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity.

Chronic cardiotoxicity of anticancer anthracyclines in the rat: role of secondary metabolites and reduced toxicity by a novel anthracycline with impaired metabolite formation and reactivity.

(1) The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues--like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755--which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. (2) DOX induced electrocardiographic and haemodynamic alterations, like elongation of QalphaT or SalphaT intervals and suppression of isoprenaline-induced dP/dt increases, which developed in a time-dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. (3) DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed approximately 60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol approximately 40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). (4) DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe-S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe-S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. (5) These results show that (i) secondary alcohol metabolites are important determinants of anthracycline-induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.

Doxorubicin-dependent reduction of ferrylmyoglobin and inhibition of lipid peroxidation: implications for cardiotoxicity of anticancer anthracyclines.

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 [Fe(IV)=O, Mb(IV)] of H(2)O(2)-activated myoglobin, a lipid oxidant likely formed in the heart during treatment with DOX. Mb(IV) was formed in vitro by reacting 100 microM H(2)O(2) with 50 microM horse heart metmyoglobin (Mb(III)). Spectral studies showed that DOX reduced Mb(IV) to Mb(III), half-maximal regeneration of Mb(III) occurring at approximately 18 microM 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 Mb(IV) through the hydroquinone moiety of its tetracyclic ring. DOX inhibited Mb(IV)-dependent peroxidation of arachidonic acid, suppressing the formation of thiobarbituric acid-reactive substances with an IC(50) of approximately 18 microM. 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 Mb(IV) reductants (ascorbate, glutathione, and ergothioneine) reached measurable IC(50)s in a micromolar range. DOX-dependent inhibition of lipid peroxidation correlated with its ability to reduce Mb(IV) to Mb(III) 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 Mb(IV) and inhibited lipid peroxidation also when H(2)O(2), Mb(III) 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 Mb(IV) to Mb(III), and cast doubts on lipid peroxidation as a causative mechanism of anthracycline-induced cardiotoxicity.