Elsevier

Biochemical Pharmacology

Volume 66, Issue 6, 15 September 2003, Pages 989-998
Biochemical Pharmacology

Anthracycline secondary alcohol metabolite formation in human or rabbit heart: biochemical aspects and pharmacologic implications

https://doi.org/10.1016/S0006-2952(03)00442-8Get rights and content

Abstract

Clinical use of the anticancer anthracyclines doxorubicin (DOX) and daunorubicin (DNR) is limited by development of cardiotoxicity upon chronic administration. Secondary alcohol metabolites, formed after two-equivalent reduction of a carbonyl group in the side chain of DOX or DNR, have been implicated as potential mediators of chronic cardiotoxicity. In the present study we characterized how human heart converted DOX or DNR to their alcohol metabolites DOXol or DNRol. Experiments were carried out using post-mortem myocardial samples obtained by ethically-acceptable procedures, and results showed that DOXol and DNRol were formed by flavin-independent cytoplasmic reductases which shared common features like pH-dependence and requirement for NADPH, but not NADH, as a source of reducing equivalents. However, studies performed with inhibitors exhibiting absolute or mixed specificity toward best known cytoplasmic reductases revealed that DOX and DNR were metabolized to DOXol or DNRol through the action of distinct enzymes. Whereas DOX was converted to DOXol by aldehyde-type reductase(s) belonging to the superfamily of aldo-keto reductases, DNR was converted to DNRol by carbonyl reductase(s) belonging to the superfamily of short-chain dehydrogenase/reductases. This pattern changed in cardiac cytosol derived from rabbit, a laboratory animal often exploited to reproduce cardiotoxicity induced by anthracyclines and to develop protectants for use in cancer patients. In fact, only carbonyl reductases were involved in metabolizing DOX and DNR in rabbit cardiac cytosol, although with different Km and Vmax. Collectively, these results demonstrate that human myocardium convert DOX and DNR to DOXol or DNRol by virtue of different reductases, an information which may be of value to prevent alcohol metabolite formation during the course of anthracycline-based anticancer therapy. These results also raise caution on the preclinical value of animal models of anthracycline cardiotoxicity, as they demonstrate that the metabolic routes leading to DOXol in a laboratory animal may not be the same as those occurring in patients.

Introduction

The anthracycline antibiotics doxorubicin (DOX) and daunorubicin (DNR) are highly effective against solid and haematologic malignancies but their use is limited by acute and chronic toxicity to the heart [1]. The acute toxicity develops immediately after initiation of anthracycline treatment and consists of transient and usually manageable arrhythmias and hypotension. In contrast, chronic cardiomyopathy develops any time after completion of cumulative anthracycline regimens and evolves into congestive heart failure, often refractory to inotropic medications. Chronic cardiotoxicity develops in a dose-dependent manner, its incidence increasing sharply at cumulative doses of ≥550 mg/m2[1], [2]. One-electron redox cycling of a quinone moiety in the tetracyclic ring of anthracyclines has been proposed to mediate cardiotoxicity induced by these drugs. In fact, several flavin-centered oxidoreductases (e.g. cytochrome P450 reductase, nitric oxide synthase, NADH dehydrogenase) catalyze one-electron reduction of this quinone to a semiquinone which readily regenerates its parent compound by reducing molecular oxygen to superoxide anion (O2radical dot) and its dismutation product hydrogen peroxide (H2O2). At the same time the semiquinone reductively releases iron from ferritin by direct or O2radical dot-mediated electron transfer. All such processes increase the cellular levels of H2O2 and Fe(II), and set the stage for formation of radical dotOH, ferryl ions and other iron–oxygen complexes able to induce oxidative damage [3], [4], [5]. While possibly explaining several aspects of the acute phase of cardiotoxicity, these mechanisms may not always explain the development of chronic cardiomyopathy. In fact, studies in laboratory animals have shown that chronic cardiomyopathy not always is accompanied by biochemical indices of oxidative damage (reviewed in Ref. [6]). Moreover, antioxidants have proven useful in delaying or preventing chronic cardiotoxicity in rodents [7] but not in dogs [8] or patients [9], [10]. In the light of these uncertainties, some investigators have proposed that chronic cardiomyopathy develops after conversion of DOX and DNR to the corresponding secondary alcohol metabolites doxorubicinol (DOXol) and daunorubicinol (DNRol) [6], [11], [12]. These metabolites are formed after two-electron reduction of a side chain C-13 carbonyl group (Fig. 1), mediated by poorly characterized cytoplasmic oxidoreductases sharing similarities with the aldo-keto reductase (AKR) and/or short-chain dehydrogenase/reductase (SDR) multigene superfamilies [13], [14]. The involvement of secondary alcohol metabolites is suggested by the following pharmacokinetic evidence: (i) the cardiac function of anthracycline-treated rodents usually declines when alcohol metabolites reach their maximum levels in the heart [6]; (ii) transgenic mice bearing cardiac-restricted overexpression of human carbonyl reductase exhibit an accelerated course of development of cardiomyopathy [12]; (iii) investigational anthracyclines with inherent resistance to carbonyl reduction induce less severe chronic cardiotoxicity in the rat [15]. From a mechanistic view point secondary alcohol metabolites are significantly less effective than their parent drugs at producing oxygen radicals, presumably because they exhibit reduced affinity for one-electron quinone reductases [16]. However, secondary alcohol metabolites are several times more potent at inactivating membrane ATPases [11], [17] and cytoplasmic aconitase/iron regulatory protein 1 [18], [19].

The possible role of secondary alcohol metabolites as biochemical determinants of chronic cardiomyopathy anticipates that the clinical use of anthracyclines could be improved by minimizing their conversion to such metabolites. As already mentioned, one approach might be to develop new anthracyclines that form less alcohol metabolites than DOX or DNR. An alternative approach might be to develop inhibitors of the reductases converting DOX and DNR to DOXol or DNRol. This latter approach clearly requires improved identification and knowledge of the function and substrate specificity of anthracycline C-13 reductases and an appraisal of possible differences between the reductases of humans and those of laboratory animals in which the protective efficacy of the inhibitors would be tested. We therefore designed experiments in which the activity and specificity of anthracycline C-13 reductases were assessed in cytosolic fractions derived from human myocardium and, for comparative purposes, in rabbit cardiac cytosol.

Section snippets

Chemicals

Doxorubicin, DOXol, DNR and DNRol were kindly provided by Pharmacia-Upjohn; stock solutions were prepared in double-distilled deionized water and shown to be stable for at least 1 month if stored at 4° in the dark. Quercetin dihydrate, kaempferol, Cibacron Blue F3G-A (CB), sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) and 85% ortho-phosphoric acid were obtained from Fluka AG; NADPH (tetrasodium salt), diphenyleneiodonium chloride (DPI), N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic

Properties of anthracycline reductase activity of human heart

Reconstitution of human cardiac cytosol with NADPH and DOX or DNR resulted in formation of DOXol or DNRol, whose production increased linearly with the incubation time (Fig. 2A). DNRol formation always exceeded that of DOXol, the DNRol/DOXol ratio ranging from 10 to 14. Under the same experimental conditions, neither microsomal nor mitochondrial fractions converted DOX or DNR to DOXol or DNRol (not shown). Heat denaturation of cytosolic fractions, omission of NADPH, or replacement of NADPH with

Discussion

Anthracycline-induced cardiotoxicity is a multifactorial process in which distinct drug metabolites or byproducts may be involved in mediating the acute and chronic phases of myocardial dysfunction [29]. As already mentioned, anthracyclines generate free radicals and the fact that cardiac-restricted catalase overexpression protects transgenic mice against anthracycline-induced cardiotoxicity supports the importance of an oxidative stress in these settings [30], [31], as does the therapeutic

Acknowledgements

We are grateful to Professor Ernesto D’Aloia (Department of Forensic Medicine of the Catholic University School of Medicine) for providing myocardial samples. This work was supported in part by grants from MURST 2001 and MIUR 2002 (to A.M.) and by MURST COFIN, FIRB and Center of Excellence on Aging at the University of Chieti (to G.M.).

References (47)

  • B.J. Cusack et al.

    Daunorubicinol-induced cardiac injury in the rabbit. A role for daunorubicinol

    Toxicol. Appl. Pharmacol.

    (1993)
  • D.D. Von Hoff et al.

    Daunomycin-induced cardiotoxicity in children and adults. A review of 110 cases

    Am. J. Med.

    (1977)
  • R. Danesi et al.

    Cytofluorescence localization and disposition of doxorubicin and doxorubicinol in rat cardiac tissue

    Eur. J. Cancer Clin. Oncol.

    (1988)
  • U.C. Oppermann et al.

    Heterogeneity of carbonyl reduction in subcellular fractions and different organs in rodents

    Biochem. Pharmacol.

    (1991)
  • R.B. Weiss

    The anthracyclines: will we ever find a better doxorubicin?

    Semin. Oncol.

    (1992)
  • P.K. Singal et al.

    Doxorubicin-induced cardiomyopathy

    New Engl. J. Med.

    (1998)
  • J.H. Doroshow

    Anthracycline antibiotic-stimulated superoxide hydrogen peroxide and hydroxyl radical production by NADH dehydrogenase

    Cancer Res.

    (1983)
  • G. Minotti et al.

    Role of iron in anthracycline cardiotoxicity: new tunes for an old song?

    FASEB J.

    (1999)
  • R.D. Olson et al.

    Doxorubicin cardio-toxicity: analysis of prevailing hypotheses

    FASEB J.

    (1990)
  • R.T. Dorr

    Cytoprotective agents for anthracyclines

    Semin. Oncol.

    (1996)
  • E.H. Herman et al.

    Comparison of the effectiveness of (±)-1,2-bis(2,5-deoxopiperanizyl-1-yl)-propane (ICRF-187) and N-acetylcysteine in preventing chronic doxorubicin cardiotoxicity in beagles

    Cancer Res.

    (1985)
  • S.S. Legha et al.

    Clinical and pharmacologic investigation of the effects of α-tocopherol on adriamycin cardiotoxicity

    Ann. N.Y. Acad. Sci.

    (1982)
  • C. Myers et al.

    A randomized controlled trial assessing the prevention of doxorubicin cardiomyopathy by N-acetylcysteine

    Semin. Oncol.

    (1983)
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