International
15
Journal of Cardiology, 29 (1990) 15-20
Ekfier CARD10 11198
Review
A new insight into adriamycin-induced cardiotoxicity LX. Fu, F. Waagstein and A. Hjaharson Wallenberg Lubomtory,
Division of Cardiolo~,
Sahlgren’s Hospital, GZkborg, Sweden
(Received 7 March 1990; revision aaxptcd 24 April 1990)
Key words: Adriamycin; Cardiotoxicity; Mechanism; Mitochondria; Cardiolipin; Free radical; Energy metabolism; Calcium overloading
Introduction Adriamycin is an anthracycling antibiotic produced by the fungus Streptomyces peucetius var. caesius. Ushered in with tremendous optimism in the last ten years, adriamycin has become an important part of the combined modality treatment of a variety of human malignancies, including both hematological and solid tumors such as histological subtypes of lung cancer, breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, thyroid carcinomas, salivary gland tumors, prostate carcinoma, sarcomas, acute leukemia, and both Hodgkin’s and non-Hodgkin’s lymphomas. With the recognition of .the potent anticancer activity of adriamycin has also come an appreciation of its toxicities, the most serious of which affects the heart. It shows a clinically important restriction to long-term treatment because it can induce a specific cardiotoxicity which is cumulative, dose dependent (total cumulative dose tolerated being 555 mg/d body surface) and potentially a life threatening form of congestive cardiomyopathy. In humans, a growing number of investigations have shown nonspecific electrocardiographic Correspondence to: Fu Liang-Xiong, M.D., WaIlenberg Laboratory, Sahlgren’e Hospiral, CNteborg University, 5-413 45 c3L)t&org, Sweden.
0167-5273/90/$03.50
0 1990 Ekvier
changes, characteristic changes in both nuclear and cytoplasmic structure, decreased ejection fraction, and congestive heart failure with increased mortality. Animal studies have demonstrated altered nuclear and cytoplasmic structure, increased coronary resistance, altered biochemistry, evidence of left ventricular failure, and increased mortality following adriamycin administration. Adriamycin cardiomyopathy is associated with chronic, rather than acute, drug administration and is both progressive and irreversible. The manifestation of cardiomyopathy is often delayed following cessation of adriamycin and occurs at ‘a time when adriamycin has been eliminated from the body. These clinical observations suggest that a biochemical mechanism more complex than direct redox cycling of adriamycin in the heart is likely to be involved in the pathogenesis of adriamycin cardiomyopathy [l]. The biochemical mechanism of adriamycin cardiotoxicity, however, remains a heated debate, although evidence is accumulating in support of several hypotheses. Adriamycin exposure may cause cellular damage via lipid peroxidation, interaction of adriamyti into nuclear and mitochondrial deoxyribonucleic acid, celhtlar calcium overloading, bioreductive activities leading to the formation of oxygen free radicals and allcylating species, and dysfunctions of mitochondria [2,3].
Science Publishers B.V. (Biomedical Division)
16
The ultimate purpose of this review is to highlight the recent plethora of information and apply this knowledge to the possible biochemical mechanisms involved in adriamycin-induced cardiotoxicity. Interaction with cardiolipin It has been shown that adriamycin binds to cell membrane and alters membrane function at or below the concentrations that affect deoxyribonucleic acid function. So far, a growing body of investigations has been undertaken to demonstrate the interaction of adriamycin with the inner membrane of heart mitochondria with consequent impairment of mitochondrial function and stimulation of free radical formation. These findings suggest a new mechanism whereby interaction of adriamycin with cardiolipin contributes to specific adriamycin cardiomyopathy. Adriamytin has been demonstrated to be able to react stoichiometrically with the negatively charged cardiolipin, a phospholipid of the mitochondrial inner membrane where it constitutes some 25% of the total lipid phosphorus and is the most plentiful in the mitochondria-rich heart, and to form an electrostatic charge-charge complex. Cardiolipin is believed to play a role in the functions of mitochondrial oxidative substrate translocators. The formation of strong complexes between adriamycin and cardiolipin results in non-oxidative inactivation of the electron transfer chain. So far, the possible mechanisms for adriamycin-induced cardiotoxicity have been proposed on the basis of interaction of mitochondrial respiratory chain with cardiolipin, since complexes I (nicotinafnide-adenine dinucleotide dehydrogenase), III (cytochrome C reductase) and IV (cytochrome C oxidase) are known to require cardiolipin in their immediate environment in order to maintain maximal enzymatic activities [4]. Adriamycin bound to the mitdchondrial membrane can also act as an electron carrier between nicotinamdie-adenine dinucleotide and cytochrome C. For instance, pyruvate is generally accepted to be an important source of energy in the cardiac mitochondria. The transport of pyruvate in mitochondria is mediated by a specific transporting system. The isolation of
the pyruvate carrier molecule and reconstitution of its transporting activity in the liposomes have been shown to be dependent upon the cardiolipin. Very recently, it has been found that the change in the activity of pyruvate translocator in thyroidhormone treated rat mitochondria was associated to parallel change in the cardiolipin content of mitochondrial membrane [S]. Such research has been encouraged by Paradies and Ruggiero [6], who have shown that the inhibition of pyruvate transport by adriamycin which may be responsible, in addition to other factors, for adriamycin cardiotoxicity may involve an interaction of adriamycin with the cardiolipin present in the lipid microenvironment surrounding the pyruvate carrier molecule in the mitochondrial membrane [7,8]. Interestingly, Sol&i and others [9-121 have shown, for the first time, that the in-vitro adriamycin binding to mitochondria can in fact be antagonized by the biologically active spermine, a widely distributed polyamine in mammalian tissue which can undergo similar electrostatic interactions with anionic membrane phospholipids (acidic phospholipids). It does indicate the existence of competition between adriamycin and spermine for both binding to the inner mitochondrial membrane and interaction with cardiolipin. Formation of free radicals Adriamycin-induced cardiotoxicity clearly involves damage to cardiac mitochondria and two main causes for this damage have been proposed: oxidative damage by oxygen free radicals and hydrogen peroxide, generated by redox cycling of adriamycin with mitochondrial nicotinamideadenine dinucleotide dehydrogenase and microsomal nicotinamide-adenine dinucleotide phosphate-cytochrome P-450 reductase (the two main intracellular sites of adriamycin reduction to the semiquinone and subsequent redox cycling with molecular oxygen); Non-oxidative inactivation of the electron transfer chain by formation of an electrostatic complex between adriamycin and cardiolipin (see above). Adriamycin is enzymatically bioactivated through one-electron reduction to free radical intermediate (semiquinone form) at complex I of the
17
mitochondrial electron transfer chain with subsequent production of oxy-free radicals by the redox cycling of semiquinone radical in the presence of molecular oxygen. It has been proposed that hydroxyl radical especially may exert major injuries to cardiac mitochondria (membrane lipid peroxidation, subsequent rigidifications of the enzymatic complexes), react with cellular deoxyribonucleic acid to induce deoxyribonucleic acid damage and oxidize certain functional proteins at thiol groups. Damage to these multiple sites may ultimately lead to cell death [13-191. Adriamycin can stimulate free radical-initiated processes such as lipid peroxidation in vitro [20,21]. The relevance of such activities in vivo in regard to adriamycin-induced cardiotoxicity is less certain. Myers et al. have demonstrated the formation of malondialdehyde, a breakdown product of lipid peroxides, in hearts of mice treated acutely with a single high dose of adriamycin [22]. This finding indicated that lipid peroxidation may be involved in the cardiotoxicity of adriamycin. Furthermore, Thayer has reported that rats chronically treated with adriamycin had relatively high amounts of lipid endoperoxides and hydroperoxides in the serum, associated with the serum lipoproteins, providing a clear indication for the occurrence of lipid reaction in vivo as a consequence of adriamycin treatment [23,24]. The different types of lipid peroxides-related compounds found in serum, as compared with tissue, may be an indication for the operation of membrane “repair” processes. Therefore, lipid peroxides may be selectively cleaved from membranes and exported to the serum where, as unusual and apparently non-metabolizable species, they might accumulate in storage lipid forms such as the triglycerides of serum lipoproteins. As shown by Thayer, serum triglycerides are also greatly elevated in adriamytin-treated rats [25]. Such kind of oxygen-dependent lipid peroxidation reaction has been recognized to be dependent upon trace amounts of iron or copper [26,27]. So far, it is understood that adriamycin binds strongly to iron and copper: ferri + 3 adriamycin --, ferri(adriamycin)3 and copper + 2 adriamycin --, copper(adriamycin),. The bound ferri or copperadriamycin complexes would be located where it
could damage the enzyme in situ through formation of reactive oxy-free radicals. It has been shown that ferri reduction occurs with the formation of an oxidized adriamycin radical and ferro chelate reacts with oxygen to yield superoxide anion or hydrogen peroxide: adriamycin - + ferri-adriamytin + ferro-adriamycin + adriamycin; oxygen + ferri-adriamycin + ferro-adriamycin + oxygen; 2 oxygen + 2 hydrogen + oxygen + hydrogen peroxide + oxygen. Additionally, it was recently shown that adriamycin is reduced to a semiquinone form at complex I of the mitochondria electron transfer chain. The redox cycling of the semiquinone radical leads to the formation af superoxide anion, hydrogen peroxide and hydroxyl free radical [28,29]. It is of interest to notice the peculiar sensitivity of the heart to free radical damage because of a less developed antioxidant defence system. Attempts to minimize adriamycin cardiotoxicity assumed that the antitumor efficiency of the adriamycin can be dissociated from its toxic side effects. Different approaches exist aiming to increase adriamycin therapeutic index: simultaneous injection of adriamycin with a specific antibody [30], injection of adriamycin encapsulated in various types of liposomes [31], association of adriamycin with deoxyribonucleic acid [32], design of adriamycin derivatives based upon the biochemical understanding of the cardiotoxicity and use of free radical scavengers [33]. Encouragingly, Praet et al. have reported that a new class of free radical scavenger-AD,, has been shown to exhibit free radical trapping properties, hence capable of reducing adriamycin cardiotoxicity due to free radical production, and to stabilize free radicals as a consequence of its capto-dative properties [28]. Besides, since mitochondria are, with the sarcoplasmic reticulum, the major sites of productions of free radicals in presence of adriamycin, it is of importance to display the efficiency of AD, against heart mitochondria toxicity of adriamycin.
Alterations of energy metabolism Since abnormal mitochondrial structure has characteristically been observed in the setting of
18
adriamycin administration in humans [34,35] and animals [36], it has been suggested that interference with mitochondrial function may be of central importance in the etiology of adriamytin-induced cardiotoxicity. As stated above, adriamycin can cause both oxidative damage and non-oxidative damage to cardiac mitochondria. The relative proportions of each form of damage are determined by the adriamycin concentration employed. It has been shown that nicotinamideadenine dinucleotide hydrogenase activity de creased by 70% following a 15 min incubation of submitochondrial particles with nicotinamide adenine dinucleotide and 50 PM adriarnycin. nicotinamide-adenine dinucleotide Likewise, oxidase activity decreased by 80% following aerobic incubation with nicotinamide-adenine dinucleotide and 50 pM adriamycin for two hours. Such losses of activity were dependent upon both nicotinamid~adenine dinucleotide and oxygen. At concentrations greater than 50 FM, adriamycin caused a nicotinamide-adenine dinucleotideindependent apparent increase in nicotinamideadenine dinucleotide dehydrogenase activity. Incubation with more than 50 PM adriamycin also caused significant inactivation of nicotinamideadenine dinucleotide oxidase, succinate dehydrogenase, succinate oxidase and cytochrome C oxidase [13,37]. These observations indicate that cardiac mitochondrial toxicity of adriamycin probably involves initial oxidative damage to nicotinamide-adenine dinucleotide dehydrogenase, with gradual loss of nicotinamide-adenine dinucleotide oxidase activity during prolonged exposure, and the nicotinamide-adenine dinucleotide oxidase and succinate oxidase impairments may be results of cardiolipin sequestrations by adriamycin since cardiolipin is required for normal complex I, III and IV activities. Significantly, Johnson et al. have reported that treatment of cultured myocytes with 10 pM adriamycin re sulted in intracellular concentration of 2 mM following 30 min incubation [38]. Such research is extremely important and worthwile because: the concentration of adriamycin used (10 $W) closely matches peak plasma concentrations observed in the clinical use of adriamycin; and mitochondrial membranes, as major targets of adriamycin toxicchronic
ity, may achieve particularly high adriamycin concentrations under such conditions because of the affinity of adriamycin for cardiolipin. Up to now, the link between acute adriamycininduced cardiotoxicity and chronic adriamycin cardiotoxicity (finally becoming adriamycin cardiomyopathy) is less well established. It is assumed that an entirely different process is responsible for the acute condition. During acute adriamycin exposure, alterations in cardiac intracellular high energy phosphate metabolism have been detected by virtue of phosphorus-31 nuclear magnetic resonance [39-411. Pelikan et al. [42] recently have demonstrated that at an adriamycin dose which clearly induces reduction in intracellular high energy phosphate and ventricular dysfunction, there was no detectable reduction in mitochondrial oxidative phosphorylation, which implies that the mechanism of acute adriamycininduced cardiotoxicity must be the consequence of something other than a primary defect in mitochondrial high energy phosphate metabolism. Since more than 90% of adenosine triphosphate produced by metabolism is generated by the Kreb’s cycle and oxidative phosphorylation [43] it is quite reasonable that the adriamycin-induced decline in the high energy phosphate might be due to a basic defect in mitochondrial metabolism initiated by adriamycin. Even if such a defect is minimal, at such high rates of turnover the energy metabolism might eventually progress into a supply/demand imbalance reflected by a decline in the high energy phosphate. In addition, calcium overloading is involved in the adriamycin-induced cardiotoxicity. So far, it is considered that adriamycin has a dual effect on the slow calcium channels of the myocardium, dependent upon the adriamycin concentration [44]. At high concentration adriamycin depresses the slow calcium channels and at low concentration it potentiates the currents. Since calcium accumulation occurs in both circumstances, but adenosine triphosphate concentration is only depressed in the former high concentration of adriamycin, it appears that depletion of adenosine triphosphate eventually depresses the slow calcium channels at high adriamycin concentration. Adriamycin-induced impairments of slow caIcium channels may
19
be due to oxidation of lipids essential membrane surrounding such channels.
to the
Conclusions Taken as a whole, although the picture that emerges from studies on adriamycin-induced cardiotoxicity is complicated and many efforts have been directed toward elucidating the basic biochemical mechanisms responsible for mediating adriamycin cardiotoxicity, it remains to be clarified. So far, to our knowledge, the interaction of adriamycin with mitochondrial cardiolipin, formations of adriamycin semiquinone and oxygen free radicals, activation of lipid peroxidation, and alteration of mitochondrial energy metabolism are recognized to play predominant roles in adriamytin-induced cardiotoxicity, especially when these multiple mechanisms function as a whole. In addition, adriamycin can result in myocardial intracellular calcium overloading which may also contribute to its cardiotoxicity. What, then, is the future for adriamycin in clinical oncological practice? Despite its unique and formidable cardiotoxicity, the adriamycin still has an extraordinarily broad spectrum of antitumor activity, and it will therefore continue to be important in cancer chemotherapy during the foreseeable future. Nevertheless, there is reason to believe that safer compounds and better clinical approaches may be in the offing. As growing laboratory evidence suggests that the mechanisms of tumor-cell killing and host tissue cardiotoxicity may be sufficiently different to be clinically dissociated, given the limitations of resources, it will be crucial to apply the biochemical understanding to human adriamycin cardiotoxicity. Furthermore, therapeutic approaches to adriamycin cardiotoxicity will be the current major topic of interest. For prospects, a rapid progress in knowledge about biochemical mechanisms of adriamycin cardiotoxicity has begged more questions to be resolved in the near future.
Acknowledgements This work was supported by the Swedish Medical Research Council and the Swedish National Association against Heart & Chest Diseases.
References 1 Thayer WS. Investigation of the role of serum lipoproteinassociated peroxides in adriamycin cardiotoxicity. Biochem Pharmacol 1989;38:1923-1929. 2 Haq MM, Legha SS, Choksi J, et al. Doxorubicin-induced congestive heart failure in adults. Cancer 1985;56:13611365. 3 Pehkan PC, Weisfeldt ML, Jacobus WE, Miceli MV, Bulkley BH, Gerstenblith G. Acute doxorubicin cardiotoxicity: functional, metabolic, and morphologic alterations in the isolated, perfused rat heart. J Cardiovasc Pharmacol 1986;8:1058-1066. 4 Fry M, Green DE. Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain. J Biol Chem 1981;256:1874-1880. 5 Paradies G, Ruggiero FM. Effect of hyperthyroidism on the transport of pyruvate in rat-heart mitochondria. Biocbim Biophys Acta 1988;935:79-86. 6 Paradies G, Ruggiero FM. The effect of doxorubicin on the transport of pyruvate in rat-heart mitochondria. Biochem Biophys Res Comrmm 1988;156:1302-1307. 7 Goormaghtigh E, Ruysschaert JM. Evidence of a convalent linkage in the adriamycin-cardiolipin complex induced by adriamycin-mediated electron transport. Res Commun Chem Path01 Pharmacol1983;42:149-152. 8 Cheneval D, Muller M, Carafoli E. The mitochondrial phosphate carrier reconstituted in liposomes is inhibited by doxorubicin. FEBS Lett 1983;159:123-126. 9 Solaini G, Landi L, Pasquali P, Rossi CA. Protective effect of endogenous coenzyme Q on both lipid peroxidation and respiratory chain inactivation induced by an adriamyciniron complex. Biochem Biophys Res Commun 1987;147:572-580. 10 Young RC, 0~01s RF, Myers CE. The anthracycline antineoplastic drugs. N Engl Med 1981;305:139-153. 11 Solaini G, Tadolini B. Spermine binding to submitochondrial particles and activation of adenosine triphosphate. Biochem J 1984;218:495-499. 12 Solaini G. Spermine antagonizes the binding of adriamycin to the inner membrane of heart mitochondria. Biochem Biophys Res Commun 1989;159:791-798. 13 Marcillat 0, Zhang Y, Davies RJ. Oxidative and nonoxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin. Biochem J 1989;259:181-189. 14 Nohl H, Jordan W. OH generation by adriamycin semiquinone and H,Or: an explanation for the cardiotoxicity of anthracycline. Biochem Biophys Res Commun 1983;114: 197-205. 15 Doroshow JH. Effect of anthracycline antibiotics on oxygen free radical formation in rat heart. Cancer Res 1983;43:460-472. 16 Davies RJ, Doroshow JH, Ho&stein P. Mitochondrial NADH dehydrogenase-catalyxed oxygen radical production by adriamycin, and the relative inactivity of 5-iminodaunorubicin. FEBS Lett 1983;153:227-230.
20 17 Doroshow JH. Anthracycline antibiotics-stimulated superoxide, hydrogen peroxide, and hydroxyl radical production by NADH dehydrogenase. Cancer Res 1983;43:4543-4551. 18 Goormaghtigh E, Brasseur R, Ruysschaert JM. Adriamycin inactivates cytochrome C oxidase by exclusion of the enzyme from its cardiohpin essential environment. Biochem Biophys Res Commun 1982;104:314-320. 19 Goormaghtigh E, Pollakis G, Ruysschaert JM. Mitochondrial membrane modifications induced by adriamycin-mediated electron transport. Biochem Pharmacol 1983;32:889-893. 20 Mimnaugh EG, Trush MA, Bhatnagar M, Gram TE. Enhancement of reactive oxygen-dependent mitochondrid membrane lipid peroxidation by the anticancer drug adriamycin. Biochem Pharmacol 1985;34:847-856. 21 Mimnaugh EG, Kennedy KA, Trush MA, Sinha BK. Adriamycin-enhanced membrane lipid peroxidation in isolated rat nuclei. Cancer Res 1985;45:3296-3304. 22 Myers CE, McGuire WP, Liss RI-I, Ifrim I, Grotzinger K, Young RC. AdriamycinThe role of lipid peroxidation in cardiac toxicity and tumor response. Science 1977;197: 165-167. 23 Thayer WS. Evaluation of tissue indicators of oxidative stress in rats treated chronically with adriamycin. Biochem Pharmacol1988;37:2189-2194. 24 Thayer WS. Serum lipid peroxides in rats treated chronically with adriamycin. B&hem Phannacol 1984;33:22592263. 25 Thayer WS. Decreased cardiac lipoprotein Iipase activity in rats treated chronically with adriamycin. Life Sci 1985;36:635-641. 26 Gutteridge JM. Lipid peroxidation and possible hydroxyl radical formation stimulated by the self-reduction of a doxorubicin-iron (III) complex. Biochem Pharmacol 1984;33:1725-1728. 27 Myers CE, Giarma L, Simone CB, Klecker R, Greene R. Oxidative destruction of erythrocyte ghost membranes catalyzed by the doxorubicin-iron complex. Biochemistry 1982;21:1707-1712. 28 Praet M, Calderon PB, PoIIakis G, Roberfroid M, Ruysschaert JM. A new class of free radical scavengers reducing adriamycin mitochondrial toxicity. Biochem Pharmacol 1988;37:4617-4622. 29 Sinha BK, Mimnaugh EG, Rajagopalan S, Myers CE. Adriamycin activation and oxygen free radical formation in human breast tumor cells: Protective roles of ghttatbione peroxidase in adriamycin resistence. Cancer Res 1989;49:3844-3848. 30 Savaraj N, ABen LM, Sutton C, Trotter M. Immunological modification of adriamycin cardiotoxicity. Res Commun Chem Path01 Pharmacol1980;29:549-559.
31 Gabizon A, Meshover A, Barenholz Y. Comparative long term study of the toxicities of free and liposomes-associated doxorubicin in mice after intravenous adminstration. J Nat1 Cancer Inst 1986;77:459-469. 32 Trouet A, Deprez-De Campeneere D, Zenebergh A, Huloven R. Lysosomotropic cancer chemotherapy with adriamycin-DNA. In: Staquet M, Tagnon H, eds. ADM review. Ghent, Belgium: European Press Medikon, Belgium, 1975;62-69. 33 Unverferth DV, Leier CV, Balcerzak AP, Hamhn RL. Usefulness of a free radical scavenger in preventing doxorubicin heart failure in dogs. Am J Cardiol 1985;56:157-161. 34 Bristow MR, Mason JW, Billingham ME, Daniels JR. Doxorubicin cardiomyopathy: evaluation by phonocardiography, endomyocardial biopsy, and cardiac characterization. Ann Intern Med 1978;88:168-175. 35 Ferrans VJ. Overview of cardiac pathology in relation to anthracycline cardiotoxicity. Cancer Treat Rep 1978;62:955-961. 36 Taylor AL, Bulkley BH. Acute adriamycin cardiotoxicity: morphologic alterations in isolated perfused rabbit heart. Lab Invest 1972;47:459-464. 37 NohI H. Identification of the site of adriamycin-activation in the heart cell. Biochem Pharmacol 1988;37:2633-2637. 38 Muhammed H, Ramasarma T, Ramakrishna, Kurup CK. Inhibition of mitochondrial oxidative phosphorylation by adriamycin. Biochim Biophys Acta 1982;722:43-50. 39 Jackson JA, Rehr RB, Peshock R, Willerson JT, Buja LM, Nunnally RL. Phosphorus nuclear magnetic resonance. study of acute adriamycin cardiotoxicity (abstract). Circulation 1983;68:111-67. 40 Ng TC, Daugherty JP, Evanochko WT, Digemess SB, Durant JR, Glickson JD. Detection of antineoplastic agent induced cardiotoxicity by 31-P NMR of perfused rat hearts. Biochem Biophys Res Common 1983;110:339-347. 41 Vick JA, Herman EH. An isolated dog or monkey heart preparation for studying cardioactive compounds. Pharmacology 1971;6:290-299. 42 Pehkan PC, Gerstenblith G, Vandegaer K, Jacobus WE. Absence of acute doxorubicin-induced dysfunction of heart mitochondrial oxidative phosphorylation and creature kinase activities. Proc Sot Exp Biol Med 1988;188;7-16. 43 Kobayashi K, Neely JR. Control of the maximum rates of gIycolysis in rat cardiac muscle. Ciic Res 1979;44:166-175. 44 Azuma J, Sperelakis N, Hasegawa H, et al Adriamycin cardiotoxicity: possible pathogenic mechanisms. J Mol Cell Cardiol 1981;13;381-397.
15
Journal of Cardiology, 29 (1990) 15-20
Ekfier CARD10 11198
Review
A new insight into adriamycin-induced cardiotoxicity LX. Fu, F. Waagstein and A. Hjaharson Wallenberg Lubomtory,
Division of Cardiolo~,
Sahlgren’s Hospital, GZkborg, Sweden
(Received 7 March 1990; revision aaxptcd 24 April 1990)
Key words: Adriamycin; Cardiotoxicity; Mechanism; Mitochondria; Cardiolipin; Free radical; Energy metabolism; Calcium overloading
Introduction Adriamycin is an anthracycling antibiotic produced by the fungus Streptomyces peucetius var. caesius. Ushered in with tremendous optimism in the last ten years, adriamycin has become an important part of the combined modality treatment of a variety of human malignancies, including both hematological and solid tumors such as histological subtypes of lung cancer, breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, thyroid carcinomas, salivary gland tumors, prostate carcinoma, sarcomas, acute leukemia, and both Hodgkin’s and non-Hodgkin’s lymphomas. With the recognition of .the potent anticancer activity of adriamycin has also come an appreciation of its toxicities, the most serious of which affects the heart. It shows a clinically important restriction to long-term treatment because it can induce a specific cardiotoxicity which is cumulative, dose dependent (total cumulative dose tolerated being 555 mg/d body surface) and potentially a life threatening form of congestive cardiomyopathy. In humans, a growing number of investigations have shown nonspecific electrocardiographic Correspondence to: Fu Liang-Xiong, M.D., WaIlenberg Laboratory, Sahlgren’e Hospiral, CNteborg University, 5-413 45 c3L)t&org, Sweden.
0167-5273/90/$03.50
0 1990 Ekvier
changes, characteristic changes in both nuclear and cytoplasmic structure, decreased ejection fraction, and congestive heart failure with increased mortality. Animal studies have demonstrated altered nuclear and cytoplasmic structure, increased coronary resistance, altered biochemistry, evidence of left ventricular failure, and increased mortality following adriamycin administration. Adriamycin cardiomyopathy is associated with chronic, rather than acute, drug administration and is both progressive and irreversible. The manifestation of cardiomyopathy is often delayed following cessation of adriamycin and occurs at ‘a time when adriamycin has been eliminated from the body. These clinical observations suggest that a biochemical mechanism more complex than direct redox cycling of adriamycin in the heart is likely to be involved in the pathogenesis of adriamycin cardiomyopathy [l]. The biochemical mechanism of adriamycin cardiotoxicity, however, remains a heated debate, although evidence is accumulating in support of several hypotheses. Adriamycin exposure may cause cellular damage via lipid peroxidation, interaction of adriamyti into nuclear and mitochondrial deoxyribonucleic acid, celhtlar calcium overloading, bioreductive activities leading to the formation of oxygen free radicals and allcylating species, and dysfunctions of mitochondria [2,3].
Science Publishers B.V. (Biomedical Division)
16
The ultimate purpose of this review is to highlight the recent plethora of information and apply this knowledge to the possible biochemical mechanisms involved in adriamycin-induced cardiotoxicity. Interaction with cardiolipin It has been shown that adriamycin binds to cell membrane and alters membrane function at or below the concentrations that affect deoxyribonucleic acid function. So far, a growing body of investigations has been undertaken to demonstrate the interaction of adriamycin with the inner membrane of heart mitochondria with consequent impairment of mitochondrial function and stimulation of free radical formation. These findings suggest a new mechanism whereby interaction of adriamycin with cardiolipin contributes to specific adriamycin cardiomyopathy. Adriamytin has been demonstrated to be able to react stoichiometrically with the negatively charged cardiolipin, a phospholipid of the mitochondrial inner membrane where it constitutes some 25% of the total lipid phosphorus and is the most plentiful in the mitochondria-rich heart, and to form an electrostatic charge-charge complex. Cardiolipin is believed to play a role in the functions of mitochondrial oxidative substrate translocators. The formation of strong complexes between adriamycin and cardiolipin results in non-oxidative inactivation of the electron transfer chain. So far, the possible mechanisms for adriamycin-induced cardiotoxicity have been proposed on the basis of interaction of mitochondrial respiratory chain with cardiolipin, since complexes I (nicotinafnide-adenine dinucleotide dehydrogenase), III (cytochrome C reductase) and IV (cytochrome C oxidase) are known to require cardiolipin in their immediate environment in order to maintain maximal enzymatic activities [4]. Adriamycin bound to the mitdchondrial membrane can also act as an electron carrier between nicotinamdie-adenine dinucleotide and cytochrome C. For instance, pyruvate is generally accepted to be an important source of energy in the cardiac mitochondria. The transport of pyruvate in mitochondria is mediated by a specific transporting system. The isolation of
the pyruvate carrier molecule and reconstitution of its transporting activity in the liposomes have been shown to be dependent upon the cardiolipin. Very recently, it has been found that the change in the activity of pyruvate translocator in thyroidhormone treated rat mitochondria was associated to parallel change in the cardiolipin content of mitochondrial membrane [S]. Such research has been encouraged by Paradies and Ruggiero [6], who have shown that the inhibition of pyruvate transport by adriamycin which may be responsible, in addition to other factors, for adriamycin cardiotoxicity may involve an interaction of adriamycin with the cardiolipin present in the lipid microenvironment surrounding the pyruvate carrier molecule in the mitochondrial membrane [7,8]. Interestingly, Sol&i and others [9-121 have shown, for the first time, that the in-vitro adriamycin binding to mitochondria can in fact be antagonized by the biologically active spermine, a widely distributed polyamine in mammalian tissue which can undergo similar electrostatic interactions with anionic membrane phospholipids (acidic phospholipids). It does indicate the existence of competition between adriamycin and spermine for both binding to the inner mitochondrial membrane and interaction with cardiolipin. Formation of free radicals Adriamycin-induced cardiotoxicity clearly involves damage to cardiac mitochondria and two main causes for this damage have been proposed: oxidative damage by oxygen free radicals and hydrogen peroxide, generated by redox cycling of adriamycin with mitochondrial nicotinamideadenine dinucleotide dehydrogenase and microsomal nicotinamide-adenine dinucleotide phosphate-cytochrome P-450 reductase (the two main intracellular sites of adriamycin reduction to the semiquinone and subsequent redox cycling with molecular oxygen); Non-oxidative inactivation of the electron transfer chain by formation of an electrostatic complex between adriamycin and cardiolipin (see above). Adriamycin is enzymatically bioactivated through one-electron reduction to free radical intermediate (semiquinone form) at complex I of the
17
mitochondrial electron transfer chain with subsequent production of oxy-free radicals by the redox cycling of semiquinone radical in the presence of molecular oxygen. It has been proposed that hydroxyl radical especially may exert major injuries to cardiac mitochondria (membrane lipid peroxidation, subsequent rigidifications of the enzymatic complexes), react with cellular deoxyribonucleic acid to induce deoxyribonucleic acid damage and oxidize certain functional proteins at thiol groups. Damage to these multiple sites may ultimately lead to cell death [13-191. Adriamycin can stimulate free radical-initiated processes such as lipid peroxidation in vitro [20,21]. The relevance of such activities in vivo in regard to adriamycin-induced cardiotoxicity is less certain. Myers et al. have demonstrated the formation of malondialdehyde, a breakdown product of lipid peroxides, in hearts of mice treated acutely with a single high dose of adriamycin [22]. This finding indicated that lipid peroxidation may be involved in the cardiotoxicity of adriamycin. Furthermore, Thayer has reported that rats chronically treated with adriamycin had relatively high amounts of lipid endoperoxides and hydroperoxides in the serum, associated with the serum lipoproteins, providing a clear indication for the occurrence of lipid reaction in vivo as a consequence of adriamycin treatment [23,24]. The different types of lipid peroxides-related compounds found in serum, as compared with tissue, may be an indication for the operation of membrane “repair” processes. Therefore, lipid peroxides may be selectively cleaved from membranes and exported to the serum where, as unusual and apparently non-metabolizable species, they might accumulate in storage lipid forms such as the triglycerides of serum lipoproteins. As shown by Thayer, serum triglycerides are also greatly elevated in adriamytin-treated rats [25]. Such kind of oxygen-dependent lipid peroxidation reaction has been recognized to be dependent upon trace amounts of iron or copper [26,27]. So far, it is understood that adriamycin binds strongly to iron and copper: ferri + 3 adriamycin --, ferri(adriamycin)3 and copper + 2 adriamycin --, copper(adriamycin),. The bound ferri or copperadriamycin complexes would be located where it
could damage the enzyme in situ through formation of reactive oxy-free radicals. It has been shown that ferri reduction occurs with the formation of an oxidized adriamycin radical and ferro chelate reacts with oxygen to yield superoxide anion or hydrogen peroxide: adriamycin - + ferri-adriamytin + ferro-adriamycin + adriamycin; oxygen + ferri-adriamycin + ferro-adriamycin + oxygen; 2 oxygen + 2 hydrogen + oxygen + hydrogen peroxide + oxygen. Additionally, it was recently shown that adriamycin is reduced to a semiquinone form at complex I of the mitochondria electron transfer chain. The redox cycling of the semiquinone radical leads to the formation af superoxide anion, hydrogen peroxide and hydroxyl free radical [28,29]. It is of interest to notice the peculiar sensitivity of the heart to free radical damage because of a less developed antioxidant defence system. Attempts to minimize adriamycin cardiotoxicity assumed that the antitumor efficiency of the adriamycin can be dissociated from its toxic side effects. Different approaches exist aiming to increase adriamycin therapeutic index: simultaneous injection of adriamycin with a specific antibody [30], injection of adriamycin encapsulated in various types of liposomes [31], association of adriamycin with deoxyribonucleic acid [32], design of adriamycin derivatives based upon the biochemical understanding of the cardiotoxicity and use of free radical scavengers [33]. Encouragingly, Praet et al. have reported that a new class of free radical scavenger-AD,, has been shown to exhibit free radical trapping properties, hence capable of reducing adriamycin cardiotoxicity due to free radical production, and to stabilize free radicals as a consequence of its capto-dative properties [28]. Besides, since mitochondria are, with the sarcoplasmic reticulum, the major sites of productions of free radicals in presence of adriamycin, it is of importance to display the efficiency of AD, against heart mitochondria toxicity of adriamycin.
Alterations of energy metabolism Since abnormal mitochondrial structure has characteristically been observed in the setting of
18
adriamycin administration in humans [34,35] and animals [36], it has been suggested that interference with mitochondrial function may be of central importance in the etiology of adriamytin-induced cardiotoxicity. As stated above, adriamycin can cause both oxidative damage and non-oxidative damage to cardiac mitochondria. The relative proportions of each form of damage are determined by the adriamycin concentration employed. It has been shown that nicotinamideadenine dinucleotide hydrogenase activity de creased by 70% following a 15 min incubation of submitochondrial particles with nicotinamide adenine dinucleotide and 50 PM adriarnycin. nicotinamide-adenine dinucleotide Likewise, oxidase activity decreased by 80% following aerobic incubation with nicotinamide-adenine dinucleotide and 50 pM adriamycin for two hours. Such losses of activity were dependent upon both nicotinamid~adenine dinucleotide and oxygen. At concentrations greater than 50 FM, adriamycin caused a nicotinamide-adenine dinucleotideindependent apparent increase in nicotinamideadenine dinucleotide dehydrogenase activity. Incubation with more than 50 PM adriamycin also caused significant inactivation of nicotinamideadenine dinucleotide oxidase, succinate dehydrogenase, succinate oxidase and cytochrome C oxidase [13,37]. These observations indicate that cardiac mitochondrial toxicity of adriamycin probably involves initial oxidative damage to nicotinamide-adenine dinucleotide dehydrogenase, with gradual loss of nicotinamide-adenine dinucleotide oxidase activity during prolonged exposure, and the nicotinamide-adenine dinucleotide oxidase and succinate oxidase impairments may be results of cardiolipin sequestrations by adriamycin since cardiolipin is required for normal complex I, III and IV activities. Significantly, Johnson et al. have reported that treatment of cultured myocytes with 10 pM adriamycin re sulted in intracellular concentration of 2 mM following 30 min incubation [38]. Such research is extremely important and worthwile because: the concentration of adriamycin used (10 $W) closely matches peak plasma concentrations observed in the clinical use of adriamycin; and mitochondrial membranes, as major targets of adriamycin toxicchronic
ity, may achieve particularly high adriamycin concentrations under such conditions because of the affinity of adriamycin for cardiolipin. Up to now, the link between acute adriamycininduced cardiotoxicity and chronic adriamycin cardiotoxicity (finally becoming adriamycin cardiomyopathy) is less well established. It is assumed that an entirely different process is responsible for the acute condition. During acute adriamycin exposure, alterations in cardiac intracellular high energy phosphate metabolism have been detected by virtue of phosphorus-31 nuclear magnetic resonance [39-411. Pelikan et al. [42] recently have demonstrated that at an adriamycin dose which clearly induces reduction in intracellular high energy phosphate and ventricular dysfunction, there was no detectable reduction in mitochondrial oxidative phosphorylation, which implies that the mechanism of acute adriamycininduced cardiotoxicity must be the consequence of something other than a primary defect in mitochondrial high energy phosphate metabolism. Since more than 90% of adenosine triphosphate produced by metabolism is generated by the Kreb’s cycle and oxidative phosphorylation [43] it is quite reasonable that the adriamycin-induced decline in the high energy phosphate might be due to a basic defect in mitochondrial metabolism initiated by adriamycin. Even if such a defect is minimal, at such high rates of turnover the energy metabolism might eventually progress into a supply/demand imbalance reflected by a decline in the high energy phosphate. In addition, calcium overloading is involved in the adriamycin-induced cardiotoxicity. So far, it is considered that adriamycin has a dual effect on the slow calcium channels of the myocardium, dependent upon the adriamycin concentration [44]. At high concentration adriamycin depresses the slow calcium channels and at low concentration it potentiates the currents. Since calcium accumulation occurs in both circumstances, but adenosine triphosphate concentration is only depressed in the former high concentration of adriamycin, it appears that depletion of adenosine triphosphate eventually depresses the slow calcium channels at high adriamycin concentration. Adriamycin-induced impairments of slow caIcium channels may
19
be due to oxidation of lipids essential membrane surrounding such channels.
to the
Conclusions Taken as a whole, although the picture that emerges from studies on adriamycin-induced cardiotoxicity is complicated and many efforts have been directed toward elucidating the basic biochemical mechanisms responsible for mediating adriamycin cardiotoxicity, it remains to be clarified. So far, to our knowledge, the interaction of adriamycin with mitochondrial cardiolipin, formations of adriamycin semiquinone and oxygen free radicals, activation of lipid peroxidation, and alteration of mitochondrial energy metabolism are recognized to play predominant roles in adriamytin-induced cardiotoxicity, especially when these multiple mechanisms function as a whole. In addition, adriamycin can result in myocardial intracellular calcium overloading which may also contribute to its cardiotoxicity. What, then, is the future for adriamycin in clinical oncological practice? Despite its unique and formidable cardiotoxicity, the adriamycin still has an extraordinarily broad spectrum of antitumor activity, and it will therefore continue to be important in cancer chemotherapy during the foreseeable future. Nevertheless, there is reason to believe that safer compounds and better clinical approaches may be in the offing. As growing laboratory evidence suggests that the mechanisms of tumor-cell killing and host tissue cardiotoxicity may be sufficiently different to be clinically dissociated, given the limitations of resources, it will be crucial to apply the biochemical understanding to human adriamycin cardiotoxicity. Furthermore, therapeutic approaches to adriamycin cardiotoxicity will be the current major topic of interest. For prospects, a rapid progress in knowledge about biochemical mechanisms of adriamycin cardiotoxicity has begged more questions to be resolved in the near future.
Acknowledgements This work was supported by the Swedish Medical Research Council and the Swedish National Association against Heart & Chest Diseases.
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