The iron chelator Dp44mT does not protect myocytes against doxorubicin

The iron chelating agent Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were compared for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle through iron-mediated oxygen radical damage. While dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, in contrast Dp44mT synergistically increased doxorubicin-induced damage. This occurred in spite of the fact that Dp44mT quickly and efficiently removed iron(III) from its complex with doxorubicin and that Dp44mT also rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex. Electron paramagnetic resonance spin trapping was used to show that iron complexes of Dp44mT were not able to generate hydroxyl radicals, suggesting that its cytotoxicity was not due to reactive oxygen species formation. In conclusion Dp44mT is unlikely to be useful as an anthracycline cardioprotective agent.     

The oral iron chelator ICL670A (deferasirox) does not protect myocytes against doxorubicin

The oral iron chelating agent ICL670A (deferasirox) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were compared for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle through iron-mediated oxygen radical damage. While dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, ICL670A, in contrast, depending upon the concentration, synergistically increased or did not affect the cytotoxicity of doxorubicin. This occurred in spite of the fact that ICL670A quickly and efficiently removed iron(III) from its complex with doxorubicin, and rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex. Continuous exposure of ICL670A to either myocytes or Chinese hamster ovary (CHO) cells resulted in cytotoxicity while treatment of CHO cells with the ferric complex of ICL670A did not. These results suggest that ICL670A was cytotoxic either by removing or withholding iron from critical iron-containing proteins. Electron paramagnetic resonance spectroscopy was used to show that neither ICL670A nor its ferric complex were able to generate free radicals in either oxidizing or reducing systems suggesting that its cytotoxicity is not due to radical generation.     

Nadph-Cytochrome-P450 Reductase Promotes Hydroxyl Radical Production by the Iron Complex of ADR-925, the Hydrolysis Product of ICRF-187 (Dexrazoxane)

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectropho-tometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.     

Hydroxyl radical production by the iron complex of the hydrolysis product of the antioxidant cardioprotective agent ICRF-187 (dexrazoxane)

SUMMARY

Dexrazoxane (ICRF-187) is now in clinical use for the prevention of doxorubicin-induced cardiotoxicity. This cardiotoxicity is thought to be due to iron-mediated oxidative stress. Dexrazoxane may be acting through its strongly metal ion binding rings-opened hydrolysis product ADR-925 by complexing iron. Since iron-chelates are known to be able to produce hydroxyl radicals, an electron paramagnetic resonance spin trapping study was undertaken to compare the hydroxyl radical-producing ability of the ferrous-ADR-925 complex with that of the ferrous complexes of ethylenediaminetetraacetic acid (EDTA) and the tetraacid analog of ADR-925 (DAPTA). In spectrophotometric studies it was shown that the ferrous-ADR-925 complex underwent aerobic oxidation 87 and 44 times slower than the ferrous complexes of EDTA or 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (DAPTA), respectively. In spite of the much slower oxidation of the ferrous-ADR-925 complex, it was, nonetheless, equally effective in producing hydrogen peroxide-dependent spin adducts. These spin adducts were produced from the reaction of the spin trap with free hydroxyl radical (HO^.), and with a transient iron oxidant with HO^.-like reactivity. Thus, it is concluded that ADR-925 acts by either complexing free iron or iron bound to doxorubicin, and forming a soluble iron complex that is less effective at producing site-specific oxygen radical damage.     

Prevention of doxorubicin-induced damage to rat heart myocytes by arginine analog nitric oxide synthase inhibitors and their enantiomers

The clinical use of the widely used anticancer drug doxorubicin is limited by a dose-dependent cardiotoxicity. Doxorubicin can be reduced to its semiquinone free radical form by nitric oxide synthases (NOS). The release of lactate dehydrogenase (LDH) from doxorubicin-treated neonatal cardiac rat myocytes was used as a model of doxorubicin-induced cardiotoxicity. The NOS inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA) protected myocytes from doxorubicin as did their non-inhibitory enantiomers D-NAME and D-NMMA. Thus, these agents did not protect by inhibiting NOS. L-NAME, which does not act at the reductase domain of NOS, also had no effect on the production of the doxorubicin semiquinone by myocytes. Nitric oxide (NO) EPR spin trapping experiments showed that L-NAME reacted with various biological reducing agents to produce NO. Ascorbic acid was highly effective in reacting with L-NAME to produce NO, while glutathione, NADPH, and NADH were much less effective. Thus, these guanadino-substituted analogs of L-arginine likely protected through their ability to slowly produce NO by reaction with intracellular ascorbic acid. Thus, some caution must be exercised in their use. NO may exert its protective effects either by directly acting as an antioxidant or through some other NO-dependent pathway.     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

L-carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation.

Besides the well-documented effect of the chemotherapeutic drug doxorubicin on free radical generation, the exact signaling mechanisms by which it causes cardiac damage remain largely unknown and are of fundamental importance in understanding anthracycline cardiotoxicity. In this study, we describe that a 1 h treatment of isolated adult rat cardiac myocytes with doxorubicin (0.5 microM) induced DNA fragmentation associated with the classical morphological features of apoptosis observed after 7 days of culture. The doxorubicin toxicity was preceded by an increase in intracellular ceramide levels with a concurrent decrease in sphingomyelin. Anthracycline-induced ceramide accumulation resulted from the activation of a sphingomyelinase assayed under acidic conditions, an effect related to an increase in V(max). Pretreatment of cardiac myocytes with L-carnitine (200 microgram/ml), a compound known for its protective effect on cardiac metabolic injuries, was found to dose-dependently inhibit the doxorubicin-induced sphingomyelin hydrolysis and ceramide generation as well as subsequent cell death. However, L-carnitine did not protect cardiac myocytes from apoptosis induced by exogenous cell-permeant ceramide. L-carnitine pretreatment did not affect the sphingomyelinase basal activity but abolished the doxorubicin-induced increase in V(max). Moreover, in vitro studies conducted on cell extracts or with purified acid sphingomyelinase demonstrated that L-carnitine exerted a dose-dependent, sphingomyelinase inhibitory effect (through V(max) reduction). Taken together, these findings show that by inhibiting a (perhaps novel) drug-activated acid sphingomyelinase and ceramide generation, L-carnitine can prevent doxorubicin-induced apoptosis of cardiac myocytes.     

Binding of the intramitochondrial ADP and its relationship to adenine nucleotide translocation

Adriamycin under partially anaerobic conditions degrades deoxyribose with the release of thiobarbituric acid-reactive products. This reaction is seen when electrons are transferred to adriamycin by xanthine oxidase or ferredoxin reductase to form the semiquinone free radical. Under the conditions described, damage to deoxyribose was inhibited by hydroxyl radicals scavengers, catalase and iron chelators. When the ratio of iron chelator to iron salt is varied both EDTA and diethylenetriamino penta-acetic acid (DETAPAC) show stimulatory properties whereas desferrioxamine remains a potent inhibitor of all reaction.     

An electron paramagnetic resonance study of the interactions between the adriamycin semiquinone, hydrogen peroxide, iron-chelators, and radical scavengers.

Anaerobic reduction of hydrogen peroxide in a xanthine/xanthine oxidase system by adriamycin semiquinone in the presence of chelators and radical scavengers was investigated by direct electron paramagnetic resonance and spin trapping techniques. Under these conditions, adriamycin semiquinone appears to react with hydrogen peroxide forming the hydroxyl radical in the presence of chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. In the absence of chelators, a related, but unknown oxidant is formed. In the presence of desferrioxamine, adriamycin semiquinone does not disappear in the presence of hydrogen peroxide at a detectable rate. The presence of adventitious iron is therefore implicated during adriamycin semiquinone-catalyzed reduction of hydrogen peroxide. Formation of alpha-hydroxyethyl radical and carbon dioxide radical anion from ethanol and formate, respectively, was detected by spin trapping. Both the hydroxyl radical and the related oxidant react with these scavengers, forming the corresponding radical. In the presence of scavengers from which reducing radicals are formed, the rate of consumption of hydrogen peroxide in this system is increased. This result can be explained by a radical-driven Fenton reaction.     

5-Iminodaunomycin. An anthracycline with unique properties

5-Iminodaunomycin forms a 3:1 complex with Fe(III) at pH 7.4. Drug-metal complex formation is associated with a marked decline in absorbance at 548 and 593 nm and the appearance of a broad band above 625 nm. The 5-iminodaunomycin-Fe(III) complex reacts with hydrogen peroxide to yield .OH radicals. This reaction is at a maximum at a drug/iron ratio of 2:1, and the yield is far less than that obtained with the doxorubicin-iron complex. In contrast to the results with doxorubicin, the production of .OH declines markedly at high 5-iminodaunomycin/iron ratios. There is a close parallel between the formation of hydroxyl radicals and the ability of the 5-iminodaunomycin complex to nick supercoiled SV40 DNA. The suppression of both .OH and DNA damage at high 5-iminodaunomycin:iron ratios is the result of several factors. 1) The presence of DNA stimulates .OH production from the doxorubicin complex, but not 5-iminodaunomycin; 2) doxorubicin reduces its chelated Fe(III) to Fe(II), but 5-iminodaunomycin does not; 3) 5-iminodaunomycin forms such a stable drug-metal complex that solvent water and, therefore, presumably H2O2, has diminished access to the chelated iron. The affinity of 5-iminodaunomycin is such that it can quantitatively abstract iron from doxorubicin. As a result, 5-iminodaunomycin is an effective competitive inhibitor of .OH radical formation by the doxorubicin-iron complex.     

Prevention of doxorubicin-induced killing of MCF-7 human breast cancer cells by oxygen radical scavengers and iron chelating agents

This study investigated the effect of oxygen radical scavengers and iron chelating agents on the toxicity of doxorubicin for MCF-7 human breast cancer cells. Superoxide dismutase and catalase, but not the heat-inactivated enzymes, the hydroxyl radical scavenger N-acetylcysteine, and the organoselenium compound 2-phenyl-1-2-benzisoselenazol-3(2H)-one, which possesses glutathione peroxidase-like activity, significantly reduced or abolished tumor cell killing by doxorubicin. Similar protective activity was found only for those iron chelating agents capable of penetrating the tumor cell plasma membrane. These experiments suggest that an iron-dependent oxygen radical cascade contributes to the antineoplastic action of the anthracycline antibiotic doxorubicin.     

Effect of zinc on superoxide-dependent hydroxyl radical production in vitro

Trace elements play an important role in oxygen metabolism and therefore in the formation of free radicals. Whereas iron and copper are usually the main enhancers of free radical formation, other trace elements, such as zinc and selenium, protect against the harmful effects of these radicals. To investigate the different protective mechanisms of zinc on radical formation, we examined the effects of added zinc and copper on superoxide dismutase activity. We also studied the effects of copper and iron on xanthine oxidase activity and on the Haber-Weiss cycle (iron, superoxide, and hydrogen peroxide), which generates hydroxyl radicals in vitro. The hypoxanthine/xanthine oxidase radical generating system contained a variety of different physiological ligands for binding the iron. This study confirmed the inhibitory effect of copper on xanthine oxidase activity. Moreover, it demonstrated that zinc inhibited hydroxyl radical formation when this formation was catalyzed by a citrate-iron complex in the hypoxanthine/xanthine oxidase reaction. Finally, human blood plasma inhibited citrate-iron-dependent hydroxyl radical formation under the same conditions. Although trace elements seemed responsible for this antioxidant activity of plasma, it is likely that zinc played no role as a plasma antioxidant. Indeed, calcium appeared to be responsible for most of this effect under our experimental conditions.     

The displacement of iron(III) from its complexes with the anticancer drugs piroxantrone and losoxantrone by the hydrolyzed form of the cardioprotective agent dexrazoxane

Piroxantrone and losoxantrone are new DNA topoisomerase II-targeting anthrapyrazole antitumor agents that display cardiotoxicity both clinically and in animal models. A study was undertaken to see whether dexrazoxane or its hydrolysis product ADR-925 could remove iron(III) from its complexes with piroxantrone or losoxantrone. Their cardiotoxicity may result from the formation of iron(III) complexes of losoxantrone and piroxantrone. Subsequent reductive activation of their iron(III) complexes likely results in oxygen-free radical-mediated cardiotoxicity. Dexrazoxane is in clinical use as a doxorubicin cardioprotective agent. Dexrazoxane presumably acts through its hydrolyzed metal ion binding form ADR-925 by removing iron(III) from its complex with doxorubicin, or by scavenging free iron(III), thus preventing oxygen-free radical-based oxidative damage to the heart tissue. ADR-925 was able to remove iron(III) from its complexes with piroxantrone and losoxantrone, though not as efficiently or as quickly as it could from its complexes with doxorubicin and other anthracyclines. This study provides a basis for utilizing dexrazoxane for the clinical prevention of anthrapyrazole cardiotoxicity.     

Mitomycin C-induced deoxyribose degradation inhibited by superoxide dismutase. A reaction involving iron, hydroxyl and semiquinone radicals

Mitomycin C stimulates deoxyribose degradation with the release of thiobarbituric acid-reactive material under conditions of low oxygen concentration. This damage is inhibited by scavengers of the hydroxyl radical, iron chelators and the specific proteins catalase and superoxide dismutase. The reactive radical species appears to arise from a Fenton-type sequence in which iron is reduced by the mitomycin C semiquinone radical.     

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.     

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Objectives and Methods of Iron Chelation Therapy

Recent developments in the understanding of the molecular control of iron homeostasis provided novel insights into the mechanisms responsible for normal iron balance. However in chronic anemias associated with iron overload, such mechanisms are no longer sufficient to offer protection from iron toxicity, and iron chelating therapy is the only method available for preventing early death caused mainly by myocardial and hepatic damage. Today, long-term deferoxamine (DFO) therapy is an integral part of the management of thalassemia and other transfusion-dependent anemias, with a major impact on well-being and survival. However, the high cost and rigorous requirements of DFO therapy, and the significant toxicity of deferiprone underline the need for the continued development of new and improved orally effective iron chelators. Within recent years more than one thousand candidate compounds have been screened in animal models. The most outstanding of these compounds include deferiprone (L1); pyridoxal isonicotinoyl hydrazone (PIH) and; bishydroxy- phenyl thiazole. Deferiprone has been used extensively as a substitute for DFO in clinical trials involving hundreds of patients. However, L1 treatment alone fails to achieve a negative iron balance in a substantial proportion of subjects. Deferiprone is less effective than DFO and its potential hepatotoxicity is an issue of current controversy. A new orally effective iron chelator should not necessarily be regarded as one displacing the presently accepted and highly effective parenteral drug DFO. Rather, it could be employed to extend the scope of iron chelating strategies in a manner analogous with the combined use of medications in the management of other conditions such as hypertension or diabetes. Coadministration or alternating use of DFO and a suitable oral chelator may allow a decrease in dosage of both drugs and improve compliance by decreasing the demand on tedious parenteral drug administration. Combined use of DFO and L1 has already been shown to result in successful depletion of iron stores in patients previously failing to respond to single drug therapy, and to lead to improved compliance with treatment. It may also result in a “shuttle effect” between weak intracellular chelators and powerful extracellular chelators or exploit the entero-hepatic cycle to promote fecal iron excretion. All of these innovative ways of chelator usage are now awaiting evaluation in experimental models and in the clinical setting.     

Bicarbonate exacerbates oxidative injury induced by antitumor antibiotic doxorubicin in cardiomyocytes

Doxorubicin, a broad-spectrum antitumor antibiotic, causes dose-dependent cardiomyopathy and heart failure. Although the exact molecular mechanisms of cardiotoxicity are not well established, oxidative mechanisms involving doxorubicin-induced superoxide anion production have been proposed. In this study, we show that bicarbonate, a physiologically relevant tissue component, greatly amplified doxorubicin-induced cardiomyocyte injury. Bicarbonate also enhanced inactivation of aconitase, a crucial tricarboxylic acid cycle enzyme, in cardiomyocytes exposed to doxorubicin. The cell-permeable superoxide dismutase mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin, reversed doxorubicin-induced cardiomyocyte injury. Bicarbonate enhanced the inactivation of purified mitochondrial aconitase in the xanthine/xanthine oxidase system, generating superoxide. The results suggest that bicarbonate amplifies the prooxidant effect of superoxide. Bicarbonate also caused an increased loading of cardiomyocytes with doxorubicin. We conclude that the bicarbonate-mediated increase in doxorubicin toxicity is due to increased intracellular loading of doxorubicin in cardiomyocytes and subsequent exacerbation of superoxide-mediated cardiomyocyte injury.     

DNA damage is an early event in doxorubicin-induced cardiac myocyte death

Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.     

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin

1. A mixture of NADPH and ferrodoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O₂ and superoxide radical is produced. 2. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine: xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. 3. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. 4. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.