Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase.
Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase.
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发表时间：
1986-03
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通讯作者：
K. Davies;J. Doroshow
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作者：
K. Davies;J. Doroshow
在本研究中，我们已经使用牛心亚线粒体制剂（BH-SMP）证明，线粒体复合物I的一个组成部分，可能是NADH脱氢酶黄素，是蒽环类抗生素还原的线粒体位点。在正向电子传递过程中，蒽环类药物阿霉素（阿霉素）和柔红霉素仅在NADH作为底物时才作为BH-SMP的单电子受体（即还原为半醌自由基）;琥珀酸盐和抗坏血酸盐没有影响。抑制剂实验（鱼藤酮，amytal，piericidin A）表明，蒽环类化合物的还原位点位于泛醌的底物侧。阿霉素和柔红霉素半醌自由基很容易检测到ESR光谱。多柔比星和柔红霉素半醌自由基（g全等为2.004，信号宽度全等为4.5 G）与分子氧反应，可能产生O2-，完成氧化还原循环。复合物I作为蒽环类抗生素还原位点的鉴定通过使用琥珀酸盐或抗坏血酸盐作为底物的ATP激发的反向电子传递的研究证实，在抗霉素A或KCN呼吸阻滞的存在下。多柔比星和柔红霉素抑制在反向电子传递过程中NAD+还原为NADH。此外，在反向电子传递过程中，在没有添加NAD+，阿霉素和柔红霉素除了引起氧消耗由于减少分子氧（O2-）的蒽环类半醌自由基。以琥珀酸为电子源，噻吩甲酰三氟丙酮（复合物II的抑制剂）和鱼藤酮阻断氧消耗，但以抗坏血酸为电子源，只有鱼藤酮是一个有效的抑制剂。在BH-SMP正向电子传递过程中，多柔比星对NADH的氧化具有99 μ M的KM和30 nmol X min-1 X mg-1的Vmax（在pH 7.4和23 ℃下）;柔红霉素的值为71 μ M和37 nmol X min-1 X mg-1。在pH 7.2和37 ° C下的氧消耗表现出对于多柔比星为65 μ M和对于柔红霉素为47 μ M的KM值，并且对于多柔比星为116 nmol X min-1 X mg-1和对于柔红霉素为114 nmol X min-1 X mg-1的Vmax值。与这些结果形成鲜明对比的是，5-亚氨基道诺比星（一种新的蒽环类药物，具有降低的心脏毒性潜力）几乎没有或没有发生还原或与BH-SMP发生氧化还原循环的趋势。蒽环类药物通过线粒体NADH脱氢酶的氧化还原循环显示在随附的论文中（Doroshow，J.H.，和Davies，K. J. A.（1986）J.Biol.Chem.261，3068 - 3074），以产生O2-、H2O2和OH，其可能是这些抗肿瘤剂的心脏毒性的基础。
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.