Original Contribution
Analyses of the Molecular Mechanism of Adriamycin-Induced Cardiotoxicity

https://doi.org/10.1016/S0891-5849(97)00025-7Get rights and content

Abstract

The molecular basis of the adriamycin(AQ)-dependent development of cardiotoxicity is still far from being clear. In contrast to our incomplete understanding of the organ-specific mechanism mitochondria are unequivocally accepted as the locus where the molecular disorder is triggered. A growing number of reports intimate the establishment of unbalanced oxygen activation through heart mitochondria in the presence of anthraquinones. In fact, in contrast to liver mitochondria, isolated heart mitochondria have been unequivocally shown to shuttle single electrons to AQ, giving rise to O2̇− formation by autoxidizing AQ̇ semiquinones. Earlier we have demonstrated the involvement of the exogenous NADH dehydrogenase in this deleterious electron deviation from the respiratory chain. This enzyme that is associated with complex I of the respiratory chain catalyzes the oxidation of cytosolic NADH. AQ activation through isolated heart mitochondria was reported to require the external addition of NADH, suggesting a flux of reducing equivalents from NADH to AQ in the cytosol. Unlike heart mitochondria, intact liver mitochondria, which are lacking this NADH-related pathway of reducing equivalents from the cytosol to the respiratory chain, cannot be made to activate AQ to semiquinones by NADH or any other substrate of respiration. It appears, therefore, that the exogenous NADH dehydrogenase of heart mitochondria exerts a key function in the myocardial toxicogenesis of anthraquinones via oxygen activation through semireduced AQ. Assessing the toxicological significance of the exogenous NADH dehydrogenase in AQ-related heart injury requires analysis of reaction products and their impact on vital bioenergetic functions, such as energy gain from the oxidation of respiratory substrates. We have applied ESR technique to analyze the identity and possible interactions of radical species emerging from NADH-respiring heart mitochondria in the presence of AQ. The following metabolic steps occur causing depression of energy metabolism in the cardiac tissue. After one-electron transfer to the parent hydrophilic anthraquinone molecule destabilization of the radical formed causes cleavage of the sugar residue. Accumulation of the lipophilic aglycone metabolite in the inner mitochondrial membrane diverts electrons from the regular pathway to electron acceptors out of sequence such as H2O2. HȮ radicals are formed and affect the functional integrity of energy-linked respiration. The key and possibly initiating role of the exogenous NADH dehydrogenase of cardiac mitochondria in this reaction pathway provides a rationale to explain the selective cardiotoxic potency of the cytostatic anthraquinone glycosides.

Introduction

Anthraquinones are increasingly used in the treatment of various forms of cancer. However, the therapeutic use is limited by the development of a dose-dependent cardiomyopathy.[1]Although a great deal of alterations on the molecular, functional, and clinical levels were reported,1, 2, 3the basic mechanism that explains the selective susceptibility of the myocardial tissues to adriamycin (AQ) is still far from being clear. Morphological changes of heart mitochondria established during AQ treatment indicate that these organelles are affected. Many research groups have, therefore, focused their scientific interest on the role of heart mitochondria in the development of AQ-related cardiomyopathy.2, 3, 4, 5, 6, 7Most effects that were reported to result from interactions with AQ, such as inhibition of adenine nucleotide translocase[8]or AQ binding to cardiolipin, which inhibits cytochrome oxidase,[9]may occur in mitochondria from other tissues as well.

In contrast toxicological activation of AQ was found to require the addition of NADH in the case of heart mitochondria,[10]while NADH and any other respiratory substrate were ineffective when liver mitochondria were exposed to AQ. The latter observations made by Davies[7]and others2, 11led to the detection of an exogenous mitochondrial NADH dehydrogenase.12, 13This enzyme was earlier shown to be associated with the cytosolic face of the inner membrane of heart mitochondria, while liver mitochondria do not have this type of enzyme.12, 14As demonstrated in previous papers,12, 14this enzyme is able to oxidize cytosolic NADH and donates electrons to the ubiquinone pool of the respiratory chain. The strong negative redox potential of this mitochondrial redox carrier, together with its accessibility to hydrophilic compounds in the cytosol, strongly suggest a reduction of the water-soluble native form of AQ catalyzed by this enzyme in the presence of cytosolic NADH.

In line with this consideration was the finding of different research groups that mitochondria catalyze a single electron reduction of AQ at the expense of extramitochondrial NADH.10, 15Furthermore, it was universally reported that AQ̇ semiquinones (AQ̇) formed in this reaction readily autoxidize and generate O2̇− radicals.6, 16, 17

While searching for an explanation of the heart-selective toxicogenesis of AQ treatment we became interested in the sequence of toxicological events after NADH-dependent AQ activation in heart mitochondria and the role of the exogenous NADH dehydrogenase therein.

Section snippets

Materials and Methods

Glutamate, succinate, KH2PO4, K2HPO4, triethanolamine, EDTA, DETAPAC, sucrose, KCN, and acetonitrile were obtained from Merck (Darmstadt, Germany); NADH and ADP were from Boehringer (Mannheim, Germany); BSA (fraction V), L-malic acid, fumarate, catalase, and superoxide dismutase (SOD) were purchased from Sigma Chemical (Deisenhofen, Germany); and adriamycin (AQ) came from Farmitalia (Bern, Switzerland). Adriamycin aglycone (AglAQ) was prepared by acid hydrolysis according to Gewirtz and

Results

Fig. 1 indirectly elucidates the involvement of the exogenous NADH dehydrogenase of heart mitochondria in the initial activation step of AQ, giving rise to the existence of paramagnetic AQ̇ semiquinone species (AQ̇). In contrast to succinate-respiring mitochondria, the use of external NADH resulted in the generation of the one-electron reduction product of AQ. The respective ESR signal exhibited a g-value of 2.0039, which is in line with the data reported for AQ̇ semiquinones in the literature.6

Discussion

This study presents experimental evidence on a sequence of toxicogenetic events that are likely to be involved in selective cardiotoxicity of AQ. The initial step in the toxicogenetic activation of AQ glycosides, the molecular form commonly used in therapy, is the one-electron reduction to the unstable AQ̇ form. This reaction was shown to require cytosolic NADH, and heart mitochondria strongly suggesting the involvement of the catalytic activity of the exogenous NADH dehydrogenase of this type

Acknowledgements

The skillful technical assistance of W. Stamberg is gratefully acknowledged.

References (27)

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