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Chem. Res. Toxicol. 2000, 13, 414-420
Doxorubicin Metabolism and Toxicity in Human Myocardium: Role of Cytoplasmic Deglycosidation and Carbonyl Reduction Sabrina Licata,† Antonella Saponiero,† Alvaro Mordente,‡ and Giorgio Minotti*,§ Institutes of Pharmacology and Biochemistry, Catholic University School of Medicine, Rome, Italy, and Department of Drug Sciences, G. D’Annunzio University School of Pharmacy, Chieti, Italy Received January 31, 2000
The anthracycline doxorubicin (DOX) is an exceptionally good antineoplastic agent, but its use is limited by formation of metabolites which induce acute and chronic cardiac toxicities. Whereas the acute toxicity is mild, the chronic toxicity can produce a life-threatening cardiomyopathy. Studies in laboratory animals are of limited value in predicting the structure and reactivity of toxic metabolites in humans; therefore, we used an ethically acceptable system which is suitable for exploring DOX metabolism in human myocardium. The system involves cytosolic fractions from myocardial samples obtained during aorto-coronary bypass grafting. After reconstitution with NADPH and DOX, these fractions generate the alcohol metabolite doxorubicinol (DOXol) as well as DOX deoxyaglycone and DOXol hydroxyaglycone, reflecting reduction of the side chain carbonyl group, reductase-type deglycosidation of the anthracycline, and hydrolase-type deglycosidation followed by carbonyl reduction, respectively. The efficiency of each metabolic route has been evaluated at low and high DOX:protein ratios, reproducing acute, single-dose and chronic, multiple-dose regimens, respectively. Low DOX:protein ratios increase the efficiency of formation of DOX deoxyaglycone and DOXol hydroxyaglycone but decrease that of DOXol. Conversely, high DOX:protein ratios facilitate the formation of DOXol but impair reductase- or hydrolase-type deglycosidation and uncouple hydrolysis from carbonyl reduction, making DOXol accumulate at levels higher than those of DOX deoxyaglycone and DOXol hydroxyaglycone. Structure-activity considerations have suggested that aglycones and DOXol may inflict cardiac damage by inducing oxidative stress or by perturbing iron homeostasis, respectively. Having characterized the influence of DOX:protein ratios on deglycosidation or carbonyl reduction, we propose that the benign acute toxicity should be attributed to the oxidant activity of aglycones, whereas the life-threatening chronic toxicity should be attributed to alterations of iron homeostasis by DOXol. This picture rationalizes the limited protective efficacy of antioxidants against chronic cardiomyopathy vis-a`-vis the better protection offered by iron chelators, and forms the basis for developing analogues which produce less DOXol.
Introduction Doxorubicin (DOX)1 is the leading compound of a broad family of anticancer anthracyclines. Since its discovery and introduction in several investigational and approved chemotherapy regimens, DOX has contributed to improved life expectancy of countless patients affected by carcinomas, sarcomas, or lymphomas (1). Unfortunately, the clinical use of such a good drug is limited by acute and chronic toxicities to cardiac tissues. The acute toxicity * To whom correspondence should be addressed: Department of Drug Sciences, G. D’Annunzio University School of Pharmacy, Via dei Vestini, 66013 Chieti, Italy. Phone: 011-39-0871-3555237. Fax: 01139-0871-3555315. E-mail:
[email protected]. † Institute of Pharmacology, Catholic University School of Medicine. ‡ Institute of Biochemistry, Catholic University School of Medicine. § G. D’Annunzio University School of Pharmacy. 1 Abbreviations: DOX, doxorubicin, (8S)-cis-10-[(3-amino-2,3,6trideoxy-R-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione; DOXol, doxorubicinol; DOX hydroxyaglycone, doxorubicin aglycone (doxorubicinone); DOXol hydroxyaglycone, doxorubicinol aglycone (doxorubicinolone); DOX deoxyaglycone, doxorubicin deoxyaglycone (7-deoxydoxorubicinone); DOXol deoxyaglycone, doxorubicinol deoxyaglycone (7-deoxydoxorubicinolone); IRP, iron regulatory protein.
develops immediately after initiation of DOX treatment and presents mild arrhythmias and hypotension; in contrast, the chronic toxicity develops after completion of cumulative dose regimens and can produce a lifethreatening form of dilative cardiomyopathy (2). The peculiar toxicity of DOX to cardiac tissues is attributed to the action of metabolites formed inside cardiomyocytes, but the precise nature of such metabolites has remained a matter of controversy because of both conceptual and technical factors. First, studies in laboratory animals are heavily influenced by species- and strain-related differences in drug metabolism; therefore, some DOX metabolites can be detected in the heart of a given animal species or strain but not in the heart of others, making it difficult to predict which metabolite(s) would form and cause cardiotoxicity in humans (3, 4). Second, studies in cancer patients are limited by ethical or practical constraints to fine-needle endomyocardial biopsies of a sufficient number and size in the accurate characterization of DOX metabolites. Third, there are several biochemical routes potentially available to DOX; however, the mechanisms governing the access to one
10.1021/tx000013q CCC: $19.00 © 2000 American Chemical Society Published on Web 04/14/2000
Doxorubicin Metabolism and Cardiotoxicity
pathway or to the other have not been defined, nor is it known whether the acute and chronic toxicities are mediated by different levels of the same metabolite or by formation of metabolites having different structures and impacts on cardiac function (5). Finally, some DOX metabolites have redox reactivity with non-heme iron, and such reactivity may convert them back to the parent anthracycline; these metabolites would therefore go underestimated or even undetected if they reacted with iron prior to their assay (6, 7). To solve these conceptual and technical problems, we have developed an in vitro human heart system, involving the reconstitution of DOX metabolism in cytosolic fractions from myocardial samples disposed during aorto-coronary bypass grafting. Once depleted of non-heme iron and reconstituted with NADPH as an enzymatic cofactor, these fractions convert DOX to metabolites which accumulate and can be assessed accurately (6, 7). To our knowledge, this is the only available system for exploring DOX metabolism in human myocardium and for establishing possible linkages to cardiotoxicity. Here we demonstrate that human cardiac cytosol can convert DOX to a secondary alcohol metabolite (DOXol) and to a broad panel of hydroxy- or deoxyaglycones, with each metabolite having the chemical prerequisites to cause cardiotoxicity. The formation of DOXol and aglycones reflects carbonyl reduction and hydrolase- or reductase-type deglycosidation of the anthracycline molecule. Moreover, we show that DOXol and aglycones are formed by mutually exclusive mechanisms and that human myocardium can switch from deglycosidation to carbonyl reduction under conditions which reproduce the acute or chronic settings of DOX treatment, respectively. These findings shed light on the biochemical foundations of DOX toxicity in the human heart and form the basis for incorporating the role of different metabolites in a pathophysiologic sequence.
Experimental Procedures Chemicals. Doxorubicin was obtained through the courtesy of A. Suarato (Chemistry Department, Pharmacia-Upjohn, Milan, Italy). Doxorubicinol and aglycone standards were prepared as described previously (6, 8). Ammonium sulfate (ultrapure grade, [FeIII] < 0.5 ppm) was purchased from Schwarz/Mann (Cleveland, OH); other chemicals were from Sigma (St. Louis, MO). Unless otherwise indicated, all preparations and experiments were carried out in 0.3 M NaCl, carefully adjusted to pH 7.0 just prior to use. While allowing us to avoid interference of most common buffers with anthracycline reactions (9, 10), these procedures and ionic strength conditions provided net buffering capacity and produced the highest yield of DOX metabolites. Solutions were prepared with doubly distilled water which had been passed through a Milli-Q Water System (Millipore, Marlborough, MA). Trace metals were eventually removed with Chelex 100 (Bio-Rad, Richmond, CA). Collection of Human Myocardium Samples. Small samples (∼0.1 g) of normothermic beating myocardium were obtained from patients undergoing aorto-coronary bypass grafting and stored at -80 °C until they were used. All biopsy samples were collected from the lateral aspect of excluded right atrium before tying the purse string of the atrial cannulation of cardio-pulmonary bypass (6). Cytosol Preparation. Pools of 15-20 biopsy samples were processed for cytosol preparation by sequential homogenization, ultracentrifugation, and 65% ammonium sulfate precipitation of 105000g supernatants (7). To prevent secondary reactions of DOXol with iron and the resultant underestimation of the amount of such a metabolite, cytosols were depleted of non-heme
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 415 iron by treatment with 100 mM dithiothreitol/100 mM Tris-HCl/ 40 mM KCl (pH 8.9) followed by Sepharose 6B chromatography (7). Proteins were determined by the bicinchoninic acid method (11). These preparations were essentially free of contamination by mitochondrial, nuclear, or microsomal NAD(P)H oxidoreductases which would have converted DOX to oxygen-consuming semiquinones. In fact, the reconstitution of cytosol (1 mg of protein/mL) with NADPH (1 mM) and DOX (0.5 mM) resulted in very little consumption of O2 [
