Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical

In the accompanying paper (Davies, K. J. A., and Doroshow, J. A. (1986) J. Biol. Chem. 261, 3060-3067), we have demonstrated that anthracycline antibiotics are reduced to the semiquinone form at Complex I of the mitochondrial electron transport chain. In the experiments presented in this study we examined the effects of doxorubicin (Adriamycin), daunorubicin, and related quinonoid anticancer agents on superoxide, hydrogen peroxide, and hydroxyl radical production by preparations of beef heart submitochondrial particles. Superoxide anion formation was stimulated from (mean +/- S.E.) 1.6 +/- 0.2 to 69.6 +/- 2.7 or 32.1 +/- 1.5 nmol X min-1 X mg-1 by the addition of 90 microM doxorubicin or daunorubicin, respectively. However, the anthracycline 5-iminodaunorubicin, in which an imine group has been substituted in the C ring quinone moiety, did not increase superoxide production over control levels. In the presence of rotenone, initial rates of oxygen consumption and superoxide formation were identical under comparable experimental conditions. Furthermore, H2O2 production increased from undetectable control levels to 2.2 +/- 0.3 nmol X min-1 X mg-1 after treatment of submitochondrial particles with doxorubicin (200 microM). The hydroxyl radical, or a related chemical oxidant, was also detected after the addition of an anthracycline to this system by both ESR spectroscopy using the spin trap 5,5-dimethylpyrroline-N-oxide and by gas chromatographic quantitation of CH4 produced from dimethyl sulfoxide. Hydroxyl radical production, which was iron-dependent in this system, occurred in a nonlinear fashion with an initial lag phase due to a requirement for H2O2 accumulation. We also found that two quinonoid anti-cancer agents which produce less cardiotoxicity than the anthracyclines, mitomycin C, and mitoxantrone, stimulated significantly less or no hydroxyl radical production by submitochondrial particles. These experiments suggest that injury to cardiac mitochondria which is produced by anthracycline antibiotics may result from the generation of the hydroxyl radical during anthracycline metabolism by NADH dehydrogenase.     

Doxorubicin cardiotoxicity: analysis of prevailing hypotheses

Anthracyclines, such as doxorubicin and daunorubicin, are highly effective anticancer agents that produce a well-described but incompletely understood cardiac toxicity. According to a popular hypothesis, anthracyclines injure the heart by generating oxygen-centered free radicals. This free radical hypothesis, however, appears to be inconsistent with many observations, such as the frequent failure of anthracyclines at cardiotoxic doses to produce evidence of increased free radical generation. Other explanations of cardiotoxicity involve platelet-activating factor, prostaglandins, histamine, calcium, and C-13 hydroxy anthracycline metabolites. These C-13 hydroxy metabolites, on the basis of in vitro data, are considerably more potent than parent compounds as myocardial depressants and as inhibitors of ATPases of sarcoplasmic reticulum, mitochondria, and sarcolemma. Further studies will be required to determine whether metabolites or the other putative injurious agents discussed contribute substantially to the cardiomyopathy of anthracycline therapy. The hypotheses presented in this paper should provide a useful framework for subsequent investigations into the mechanisms of anthracycline cardiotoxicity.     

Anthracycline binding to synthetic and natural membranes. A study using resonance energy transfer

The binding of adriamycin and its two analogues 4'-epidoxorubicin and 4'-deoxydoxorubicin to synthetic and mitochondrial membranes was investigated by using resonance energy transfer between these drugs and two fluorescent probes, diphenylhexatriene (DPH) and tryptophan. The fluorescence of the lipid probe DPH in both types of membranes and tryptophan in mitochondria was quenched by the anthracyclines in a dose-dependent manner. In sonicated, fluid-phase dimyristoyl-L-alpha-phosphatidylcholine (DMPC) vesicles, the half-quenching concentration (K50) of adriamycin was 17 +/- 1 microM, whereas in bilayers containing a 1:1 molar ratio of DMPC to cardiolipin (CL), the value was 8 +/- 1 microM. In liver and heart mitochondria, the K50 values were 8 +/- 2 and 11 +/- 3 microM, respectively. Similar results were obtained for the other two drugs. Replacing a nonionic with an ionic medium or decreasing the pH from pH 7.7 to pH 6.9 increased the K50 value of adriamycin for DPH in DMPC/CL (1:1 molar) liposomes and in mitochondria. Higher concentrations of anthracycline were needed to quench the fluorescence of tryptophan. The results suggest that these drugs interact with both phospholipids and proteins and that the cardiotoxicity of adriamycin is unlikely to be related to the amount of drug bound to heart mitochondria.     

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.     

Carvedilol inhibits the exogenous NADH dehydrogenase in rat heart mitochondria

There are several reports on the oxidation of external NADH by an exogenous NADH dehydrogenase in the outer leaflet of the inner membrane of rat heart mitochondria. Until now, however, little was known about its physiological role in cellular metabolism. The present work shows that carvedilol (?1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-pro - panol-(2)?) is a specific inhibitor of an exogenous NADH dehydrogenase in rat heart mitochondria. Carvedilol does not affect oxygen consumption linked to the oxidation of succinate and internal NADH. It is also demonstrated that the inhibition of exogenous NADH dehydrogenase by carvedilol is accompanied by the inhibition of alkalinization of the external medium. In contrast to the addition of glutamate/malate or succinate, exogenous NADH does not generate a membrane potential in rat heart mitochondria, as observed with a TPP(+) electrode. It is also demonstrated that the oxygen consumption linked to NADH oxidation is not due to permeabilized mitochondria, but to actual oxidase activity in the inner membrane. The enzyme has a K(m) for NADH of 13 microM. Carvedilol is a noncompetitive inhibitor of this external NADH dehydrogenase with a K(i) of 15 microM. Carvedilol is the first inhibitor described to this organospecific enzyme. Since this enzyme was demonstrated to play a key role in the cardiotoxicity of anticancer drugs of the anthracycline family (e.g., adriamycin), we may suggest that the administration of carvedilol to tumor patients treated with adriamycin might be of great help in the prevention of the cardioselective toxicity of this antibiotic.     

Cytofluorescence detection of adriamycin-mitochondria interactions in isolated, perfused rat heart

The major side-effect of the anthracycline anti-tumor drug adriamycin is a specific, dose-dependent cardiotoxicity. Impairment of mitochondrial function has been suggested to play an important role in this toxicity. The present study addresses the question as to whether direct drug-mitochondria interactions occur in the isolated, perfused rat heart. To this aim, cytofluorescence microscopy experiments were performed on thin cryosections. To demonstrate the applicability of this technique it is shown that adriamycin bound to isolated rat liver and heart mitochondria can be visualized through its characteristic fluorescence. Longitudinal sections from heart tissue perfused with 50 microM adriamycin display two distinct cellular sites of drug accumulation, i.e., nuclei which exhibit very bright fluorescence and, in addition, mitochondria which become significantly labeled with the drug. The mitochondrial localization of adriamycin is confirmed independently by quantification of the drug content of the mitochondrial fraction after cell fractionation. These results are discussed in the light of the potential role of adriamycin-nuclei versus adriamycin-mitochondria interactions in the deterioration of heart performance.     

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of th respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c1 segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

Investigation of the interaction of superoxide ion with adriamycin and the possible origin of cardiotoxicity of the anthracycline anticancer antibiotics

Interaction of superoxide ion with adriamycin in an aprotic medium has been studied. It was shown that superoxide ion reacts irreversibly with adriamycin, giving a diamagnetic product (the dimer or oligomer of semiquinone) which can be reoxidized to adriamycin. This product was also obtained when adriamycin reacted with benzosemiquinone, ubisemiquinone, and the semiquinones of tocopherylquinone and vitamin K₁. It is suggested that the cardiotoxicity of adriamycin and other anthracycline anticancer antibiotics is caused by the high elcctron-attracting properties of these antibiotics, while the ability of natural quinones to reduce cardiotoxicity and to induce recovery of respiration in mitochondria is due to their interaction with the semiquinone states of the antibiotics.     

Non-Enzymic Lipid Peroxidation in Microsomes and Microsomal Phospholipids Induced by Anthracyclines

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes.

Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation.

Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system.

The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.     

Non-Enzymic Lipid Peroxidation in Microsomes and Microsomal Phospholipids Induced by Anthracyclines

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes.

Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation.

Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system.

The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.     

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c₁ segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

Reduced nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation by beef heart submitochondrial particles.

Electron transport particles (ETP) prepared from beef heart mitochondria formed malondialdehyde by NADPH-dependent lipid peroixidation in the presence of ferric ions and ADP or ATP. The reaction was inhibited by MnCl2, EDTA, or radical scavengers, but was not inhibited by p-hydroxymercuribenzoate (PHMB) or respiratory chain inhibitors. The oxidation of NADPH and oxygen consumption by ETP were activated by the addition of ferric ions and APT, and inhibited by inhibitors of lipid peroxidation. This peroxidation system was apparently different from those of liver microsomes and mitochondria as regards the effect of PHMB, optimal pH and the concentration of NADPH for half-maximal reaction velocity.     

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.     

Effects of doxorubicin‐induced cardiotoxicity on cardiac mitochondrial dynamics and mitochondrial function: Insights for future interventions

Anthracyclines is an effective chemotherapeutic treatment used for many types of cancer. However, high cumulative dosage of anthracyclines leads to cardiac toxicity and heart failure. Dysregulation of mitochondrial dynamics and function are major pathways driving this toxicity. Several pharmacological and non‐pharmacological interventions aiming to attenuate cardiac toxicity by targeting mitochondrial dynamics and function have shown beneficial effects in cell and animal models. However, in clinical practice, there is currently no standard therapy for the prevention of anthracycline‐induced cardiotoxicity. This review summarizes current reports on the impact of anthracyclines on cardiac mitochondrial dynamics and mitochondrial function and potential interventions targeting these pathways. The roles of mitochondrial dynamics and mitochondrial function in the development of anthracycline‐induced cardiotoxicity should provide insights in devising novel strategies to attenuate the cardiac toxicity induced by anthracyclines.     

Formation of superoxide radicals in isolated cardiac mitochondria: effect of adriamycin

The effect of adriamycin (doxorubicin) on superoxide radical formation in isolated rat heart mitochondria was studied by the spin trapping technique. The samples were placed into the cavity of EPR spectrometer in thin - wall gas - permeable capillary tubes, which allowed keeping the mitochondria of suspension in aerobic conditions. TIRON was used as a spin trap. We demonstrated that the rate of superoxide generation by isolated mitochondria depended radically on the presence of 1-150 microM adriamycin in incubation medium and was considerably higher than in control. The effect of adriamycin could be observed in the presence of both complex I (succinate) or complex II (glutamate and malate) substrates. The results obtained let to conclude that isolated cardiac mitochondria modified by adriamycin have a higher rate of production of superoxide radicals, which can react with spin traps not penetrating through the internal membrane.     

Adriamycin stimulated superoxide formation in submitochondrial particles.

Adriamycin (doxorubicin), an anticancer agent, stimulated the formation of superoxide in submitochondrial particles isolated from bovine heart. Superoxide formation was detected by oxygen uptake, by the cooxidation of epinephrine to adrenochrome and by the reduction of acetylated cytochrome c. These processes were sensitive to superoxide dismutase (SOD). Rotenone-insensitive oxidation of NADH by the mitochondrial respiratory chain in the presence of oxygen caused the formation of approx 4 nmol of superoxide per min/mg of protein. Adriamycin at a concentration of 400 micron stimulated the rate of superoxide formation 6-fold to 25 nmol.min-1.mg-1, but this was not a maximum rate. Approximately 50 micron adriamycin was estimated to be sufficient for obtaining one-half maximal stimulation. Hydrogen peroxide accumulated as a final reaction product. Measurements of the relative catalase activity of blood-free tissues of rabbits and rats indicated that heart contained 2 to 4% of the catalase activity of liver or kidney. An enhanced production of superoxide and hydrogen peroxide and the relatively low catalase content of heart tissue may be factors in the cardiotoxicity induced by adriamycin chemotherapy if a similar reaction occurs in vivo.     

Experience with using carminomycin in oncological clinical practice]

Carminomycin is an original antitumor antibiotic from the anthracycline group isolated at the Institute of New Antibiotics (USSR) in 1973. Pharmacological investigation of carminomycin revealed its satisfactory absorption from the gastrointestinal tract which proved to be a distinguishing property of the antibiotic as compared to other anthracyclines such as adriamycin and rubomycin. The clinical trials of carminomycin showed that it was mainly active against soft tissue sarcoma and breast cancer, lymphosarcoma, neuroblastoma, Wilms' tumor and Ewing's sarcoma in children, as well as acute leukemia. Various regimens for the antibiotic administration were applied: short-term, single and long-term. Suppression of hemopoiesis was considered as a limiting toxic effect. By the data available carminomycin had lower cardiotoxicity as compared with rubomycin and adriamycin. Development of oral carminomycin is believed promising.     

An Unusual Nad(P)H-Dependent Oi-Generating Redox System in Hepatoma 22a Nuclei

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes⁷ which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.     

Adriamycin as an inhibitor of 11 beta-hydroxylase activity in adrenal cortex mitochondria

Inhibition of 11 beta-hydroxylase activity was observed to be due to the interaction of adriamycin with adrenal cortex mitochondria. The inhibition of the enzyme was uncompetitive, with an apparent Ki of 100 microM, and was dependent upon the concentration of the drug and the time of incubation. Adriamycin increased the oxygen consumption of these mitochondria. EPR studies showed that adriamycin was reduced to a free radical semiquinone which served to shuttle electrons to oxygen, leading to an impairment in the reduction of cytochrome P450. It is suggested this may be the mechanism for the inhibitory effect of the drug on 11 beta-hydroxylase activity.     

An Unusual Nad(P)H-Dependent Oi-Generating Redox System in Hepatoma 22a Nuclei

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes⁷ which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.