Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase.

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.