Oxidative degradation of cardiotoxic anticancer anthracyclines to phthalic acids: Novel function for ferrylmyoglobin

We show that the pseudoperoxidase activity of ferrylmyoglobin (Mb ^IV) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. Mb ^IV, formed in vitro by reacting horse heart Mb^III 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/Mb^IV 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 Mb^IV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and Mb^III/H₂O₂ or hemin/H₂O₂ showed that the porphyrin radical of Mb^IV-compound I and the iron-oxo moiety of Mb^IV-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 Mb^IV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity.