Anthracycline metabolism and toxicity in human myocardium: comparisons between doxorubicin, epirubicin, and a novel disaccharide analogue with a reduced level of formation and [4Fe-4S] reactivity of its secondary alcohol metabolite.

Secondary alcohol metabolites have been proposed to mediate chronic cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines. In this study, NADPH-supplemented human cardiac cytosol was found to reduce the carbonyl group in the side chain of the tetracyclic ring of DOX, producing the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in the level of alcohol metabolite formation was observed by replacing DOX with epirubicin (EPI), a less cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the hydroxyl group at C-4 in the amino sugar bound to the tetracyclic ring (daunosamine). A similar decrease was observed by replacing DOX with MEN 10755, a novel anthracycline with preclinical evidence of reduced cardiotoxicity. MEN 10755 is characterized by the lack of a methoxy group at C-4 in the tetracyclic ring and by intercalation of 2, 6-dideoxy-L-fucose between daunosamine and the aglycone. Multiple comparisons with methoxy- or 4-demethoxyaglycones, and a number of mono- or disaccharide 4-demethoxyanthracyclines, showed that both the lack of the methoxy group and the presence of a disaccharide moiety limited alcohol metabolite formation by MEN 10755. Studies with enzymatically generated or purified anthracycline secondary alcohols also showed that the presence of a disaccharide moiety, but not the lack of a methoxy group, made the metabolite of MEN 10755 less reactive with the [4Fe-4S] cluster of cytoplasmic aconitase, as evidenced by its limited reoxidation to the parent carbonyl anthracycline and by a reduced level of delocalization of Fe(II) from the cluster. Collectively, these studies (i) characterize the different influence of methoxy and sugar substituents on the formation and [4Fe-4S] reactivity of anthracycline secondary alcohols, (ii) lend support to the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they demonstrate that the less cardiotoxic EPI and MEN 10755 share a reduction in the level of formation of such metabolites, and (iii) suggest that the cardiotoxicity of MEN 10755 might be further decreased by the reduced [4Fe-4S] reactivity of its alcohol metabolite.

The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium.

Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH-supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe-4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX-Fe(II) complexes as ultimate by-products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein-1 (IRP-1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP-1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol-dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP-1 or to reincorporate iron within new Fe-S motifs. This damage is mediated by DOX-Fe(II) complexes and reflects oxidative modifications of -SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP-1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP-1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.

Intermittent antegrade hyperkalaemic warm blood cardioplegia supplemented with magnesium prevents myocardial substrate derangement in patients undergoing coronary artery bypass surgery.

OBJECTIVE: The influence of the addition of magnesium on myocardial protection with intermittent antegrade warm blood hyperkalaemic cardioplegia in patients undergoing coronary artery surgery was investigated and compared with intermittent antegrade warm blood hyperkalaemic cardioplegia only. METHODS: Twenty-three patients undergoing primary elective coronary revascularization were randomized to one of two different techniques of myocardial protection. In the first group, myocardial protection was induced using intermittent antegrade warm blood hyperkalaemic cardioplegia. In the second group, the same technique was used except that magnesium was added to the cardioplegia. Intracellular substrates (ATP, lactate and amino acids) were measured in left ventricular biopsies collected 5 min after institution of cardiopulmonary bypass, after 30 min of ischaemic arrest and 20 min after reperfusion. RESULTS: There were no significant changes in the intracellular concentration of ATP or free amino acid pool in biopsies taken at the end of the period of myocardial ischaemia. However, the addition of magnesium prevented the significant increase in the intracellular concentration of lactate seen with intermittent antegrade warm blood hyperkalaemic cardioplegia. Upon reperfusion there was a significant fall in ATP and amino acid concentration when the technique of intermittent antegrade warm blood hyperkalaemic cardioplegia was used but not when magnesium was added to the cardioplegia. CONCLUSIONS: This work shows that intermittent antegrade warm blood hyperkalaemic cardioplegia supplemented with magnesium prevents substrate derangement early after reperfusion.