The Heart As a Target for Xenobiotic Toxicity: The Cardiac

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The heart as a target for xenobiotic toxicity: the cardiac susceptibility to oxidative stress Vera Marisa Costa, Félix Carvalho, José Duarte, Maria Lourdes Bastos, and Fernando Remião Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx400130v • Publication Date (Web): 31 Jul 2013 Downloaded from http://pubs.acs.org on August 2, 2013

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Chemical Research in Toxicology

The heart as a target for xenobiotic toxicity: the cardiac susceptibility to oxidative stress

Vera Marisa Costa1*, Félix Carvalho1, José Alberto Duarte2, Maria de Lourdes Bastos1, and Fernando Remião1 1

REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia,

Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2CIAFEL, Faculdade de Desporto, Universidade do Porto, Porto, Portugal

Abstract The heart is a target organ for oxidative stress-related injuries. Due to its very high energetic metabolic rate, the heart has the highest rate of production of reactive oxygen species, namely hydrogen peroxide (H2O2), per gram of tissue. Additionally, the heart has lower levels of antioxidants and of total activity of antioxidant enzymes when compared to other organs. Furthermore, drugs that have relevant antioxidant activity and that are used in the treatment of oxidative stress related cardiac diseases demonstrate better clinical cardiac outcomes than other drugs with similar receptor affinity but with no antioxidant activity. Several xenobiotics particularly target the heart and promote toxicity. Anticancer drugs, like anthracyclines, cyclophosphamide, mitoxantrone, and more recently tyrosine kinase targeting drugs are well known cardiac toxicants whose therapeutic application has been associated to a high prevalence of heart failure. High levels of catecholamines or drugs of abuse, namely amphetamines, cocaine, and even the consumption of alcohol for long periods of time are linked to cardiovascular abnormalities. Oxidative stress may be one common link for the cardiac toxicity elicited by these compounds. We aim to revise the mechanisms involved in cardiac lesions caused by the above mentioned substances specially focusing in oxidative stress related pathways. Oxidative stress biomarkers can be useful in the early recognition of cardiotoxicity in patients treated with these drugs and aid to minimize the setting of cardiac irreversible events. 1 ACS Paragon Plus Environment


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Corresponding Author: Vera Marisa Costa (E-mail: [email protected]) Departamento de Ciências Biológicas, Laboratório de Toxicologia Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal Telephone 00351-22 0428796

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TABLE OF CONTENT GRAPHIC TOXIC METABOLITES REDOX CYCLE - Anthracyclines - Catecholamines - MDMA (catechol metabolites)

↓ ANTIOXIDANT DEFENSES - Anthracyclines - Ethanol (acetaldehyde) -Mitoxantrone -Mitoxantrone -Cyclophosphamide - MDMA (catechol metabolites)

MITOCHONDRIAL TOXINS RELEASE OF CATECHOLAMINES - Amphetamines

- Anthracyclines

Oxidative stress

-Mitoxantrone

- Cocaine

BLUNT SURVIVAL PATHWAYS - Trastuzumab

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3 Mitochondrial dysfunction


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Abbreviations

H2O2 – Hydrogen peroxide HO● – Hydroxyl radical ONOO− – Peroxynitrite HOO● – Peroxyl radical O2●_ – Superoxide anion radical ADH – Alcohol dehydrogenase ADP – Adenosine diphosphate ALDH – Aldehyde dehydrogenase ATP – Adenosine triphosphate BNP – Brain natriuretic peptide cTnI – Cardiac troponin I cTnT – Cardiac troponin T CK-MB – Creatine kinase-MB CNS – Central nervous system GPX – Glutathione peroxidase GR – Glutathione reductase GSH – Reduced glutathione GSSG – Oxidized glutathione disulfide GST – Glutathione-S-transferase i.p. – Intraperitoneal i.v. – Intravenous HER – Human epidermal growth factor receptor


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LVEF – Left ventricular ejection fraction MAPK – Mitogen-activated protein kinase MAO – Monoamine oxidase MDMA – 3,4-Methylenedioxymethamphetamine NADH – Nicotinamide adenosine dinucleotide NADPH – Nicotinamide adenosine dinucleotide phosphate ●

NO – Nitric oxide

NOS – ●NO synthase NT-proBNP – N-terminal prohormone of brain natriuretic peptide RNS – Reactive nitrogen species ROS – Reactive oxygen species SOD – Superoxide dismutase



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1. Introduction

The heart is an extremely hard working organ as it pumps on average 10 tons of blood with about 100,000 beats per day. Its mechanical and electrophysiological functions require efficient energy supply and high energy pools. β-Oxidation of fatty acids coupled with mitochondrial oxidative phosphorylation lead to a relative efficient energy formation in the heart. These processes involve redox mechanisms where oxygen plays a major role, therefore ensuing the formation of significant amounts of reactive oxygen species (ROS).1 An important area of investigation addresses the sources and effects of ROS and reactive nitrogen species (RNS) in heart diseases and the factors responsible for their regulation. At low levels, ROS and RNS contribute to a basal endogenous redox buffering environment that reversibly interacts with specific cellular targets, thus creating conditions towards optimal performance.2 ROS have several subtle effects in the remodeling or failing heart that involve specific redox-regulated signaling pathways.3-5 Low ROS levels have been shown to repress the activity of transcription factors, specifically GATA binding protein 4 (GATA4) and nuclear factor of activated T cells (NFAT), which are involved in the development of cardiac hypertrophy, for example.6 The heart is particularly sensitive to these redox sensitive mechanisms as they are useful for the adaptation towards the body’s demands. Typically, sulfhydryl (-SH) groups of cysteine residues are potential targets for redox modification of proteins. The formation (or breaking) of disulfide bonds affects the structure and function of several cardiac proteins including cardiac ion channels, pumps, and transporters.2 The specificity of redox modulation depends on many factors including the redox state of the intracellular (micro-) environment, and the concentration and nature of the redox active molecules, being ROS and RNS considered the major biologically relevant redox active molecules.1 At high concentrations, however, redox active species are capable of irreversibly modifying and damaging lipids and proteins or compromising the function of enzyme or transporters, as seen in pathologic states like ischemia-reperfusion or heart failure.1, 2 When a redox imbalance occurs, it is, in general, named oxidative stress. Oxidative stress is known to correspond to a disturbance in the pro-oxidant/antioxidant balance in favor of the former, resulting in potential damage.7 The heart has low amounts of antioxidants when compared to other organs; however, it produces a high amount of basal pro-oxidants, which makes it particularly susceptible to oxidative stress damage. Thus, oxidative stress occurs by decreases in antioxidant levels, increases in


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production of ROS and/RNS or both. Briefly, we will address some unique characteristics of cardiac antioxidant defenses and reactive species formation. In spite of the high basal rate of reactive species formation, the heart has several antioxidant defense limitations, when compared to liver or kidney, for instance. 8 Glutathione [together with the glutathione peroxidase (GPX)/reductase system, glutathione reductase (GR) and glutathione S-transferases (GST)], the thioredoxin system and the enzymes superoxide dismutase (SOD) and catalase constitute the key elements for the cellular defense against redox injuries.1, 2 These mechanisms aim to protect cells by maintaining superoxide anion radical (O2●−) and H2O2 at low levels 9 and therefore reducing substantially the formation of the highly deleterious peroxynitrite (ONOO−) or hydroxyl radical (HO●). The dismutation of O2●− by cytosolic copper/zinc and mitochondrial manganese containing superoxide dismutases (CuZnSOD and MnSOD, respectively) leads to the formation of the deleterious and cellular permeable H2O2. Up to 70% of SOD activity in the heart and up to 90% of the total SOD activity in cardiac myocytes is mitochondrial MnSOD.10, 11 The remaining SOD consists primarily of cytosolic CuZnSOD. MnSOD has a crucial role in controlling mitochondrial O2●− levels, which are formed during oxidative phosphorylation10 and the enzyme levels were found to be approximately 8.4 units in the human heart.12 Catalase and GPX are the enzymes involved in the detoxification of H2O2, being catalase the most relevant.1 In the heart, catalase activity is low (approximately 31 µmol of H2O2/min.mg of protein in rat heart 13) and it is mainly concentrated in cytosolic peroxisomes. Its main function is the detoxification of H2O2 produced as a result of peroxisome aerobic dehydrogenase reactions.14 The importance of catalase in providing protection against extra peroxisome H2O2 is not clear. On the other hand, GPX is mainly present in cytosol, but also in mitochondria (10%), endoplasmic reticulum, and nuclei. The elimination of H2O2 by GPX is accomplished with the concomitant oxidation of reduced (GSH), thus imposing both an oxidative- and an energetic stress (through the expense of nicotinamide adenosine dinucleotide phosphate - NADPH) on the cell.15 The increase in oxidized glutathione disulfide (GSSG) levels can promote the efflux of GSSG from the cell, through multidrug resistance proteins or the formation of mixed disulfides in cellular proteins.16, 17 A significant number of proteins involved in signaling have critical thiols.2, 18 GSH-conjugating enzymes, GSTs, are present in different subcellular compartments including cytosol, mitochondria, endoplasmic reticulum, nucleus, and plasma membrane. GSTs catalyze the conjugation of GSH with electrophilic xenobiotics, or endogenous unsaturated aldehydes, quinones, epoxides, and peroxyl radical (HOO●).9 7 ACS Paragon Plus Environment


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In the human right atria GST activity was found to be 45.7 ± 3.41 nmol of substrate conjugated in the presence of 1-chloro-2,4-dinitrobenzene/(mg.protein.min).19 Another glutathione-dependent enzyme is GR that reduces GSSG by using NADPH as the reducing cofactor.9 The levels of GR in the human heart are moderate [17.1 ± 1.14 nmol NADPH oxidized/(mg of protein.min) in human right atria]19 when compared to the values found in the liver.19, 20 The ubiquitously expressed thiol-reducing systems also include the thioredoxin reductase/thioredoxin system, glutaredoxin, and reduced glutathione (GSH).21 In many mammalian cells, including cardiomyocytes, GSH is considered the major cytosolic redox buffer. Under normal physiological conditions, glutathione is mainly in the reduced form and the ratio of GSH to GSSG should be greater than 10.22 The maintenance of a high cytosolic GSH/GSSG ratio is a critical factor for cellular antioxidant defenses and overall homeostasis. GSH can directly scavenge ROS or indirectly through the reaction catalyzed by GPX.22 When oxidative stress occurs, the GSH/GSSG ratio can decrease significantly and rapidly.23 Considerable attention has been given to the study of other non-enzymatic antioxidants, particularly the antioxidant vitamins E, A, and C in the mitigation of cardiovascular events. Vitamin E is a lipid soluble antioxidant and that acts as a potent HOO• scavenger being able to interrupt the lipid peroxidation chain reaction.24 There are significant amounts of vitamin E in the myocardial cytosolic and mitochondrial membranes.24 Vitamin C is as a water-soluble electron-transport antioxidant present in the cytosol and in the extracellular fluid.24 In vitro, it is able to scavenge a wide variety of radicals, specifically O2●−, peroxyl-derived radicals, and HO●.25 Vitamin A or retinol is described as a scavenger to some reactive species 25 as it inhibits lipid peroxidation, either alone 26 or with other vitamins.27 The scavenging ability of β-carotene, the most relevant vitamin A generating carotenoid, is also reported, although it has low antioxidant ability.28-30 Other antioxidant defenses are present in the cardiac tissue, but they will only be addressed if required in any specific section of this review. These cardiac weapons against oxidative or nitrosative stress have several action mechanisms and multiple cellular localizations; however they may be markedly insufficient even when a mild oxidative stress stimulus occurs.1 Moreover, the sources of reactive species in the heart are abundant, making the heart redox homeostasis hard to achieve.


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Within cardiac cells, reactive species are produced in multiple compartments and have several sources, including NADPH oxidases at the plasma membrane, mitochondrial oxidative phosphorylation or β-oxidation within the mitochondria and peroxissomes, the action of monoamine oxidases (MAO), p66Shc, cyclooxygenases, P450 cytochrome or xanthine oxidase. The latter (cyclooxygenases, P450 cytochrome or xanthine oxidase) are not considered to be of great relevance except in very specific heart problems and will not be addressed. Nitric oxide (•NO) can also contribute to the overall oxidative/nitrosative imbalance in the heart. Although all of these sources contribute to the overall oxidative burden, mitochondria contribute to the majority of ROS generation due to the electron leakage in the respiratory chain.31 Mitochondria density is high in the heart, constituting up to 35% of the cell’s volume in rat heart muscle cells. The mitochondrial electron transport chain reduces more than 95% of the O2 consumed by tetravalent reduction to H2O, without the formation of ROS. However, the remaining oxygen may be reduced via the univalent pathway where a small number of electrons may prematurely escape from the electron transport chain to oxygen, forming O2●−.18, 32, 33

The O2●− is produced essentially by complexes I and III of the electron transport

chain.34, 35 Furthermore, inhibition of the electron transfer chain favors ROS formation 1 and mitochondrial sites other than the inner mitochondrial membrane are capable of generating significant amounts of H2O2. In fact, MAOs catalyze the biotransformation of biogenic amines with electron transfer to O2 in the outer mitochondrial membrane.1 β-Oxidation of fatty acids is the major source of acetyl-coA in the heart, a metabolic pathway that operates both in mitochondria and peroxisomes, and is also a great source of ROS. Indeed, the first step in peroxisome fatty acid-oxidation is directly coupled to molecular oxygen to produce H2O2, which contributes to oxidative injury if not dealt by cellular antioxidant enzymes.24 NADPH oxidases are relevant sources of ROS in cardiomyocytes and other heart cells, namely vascular cells.36, 37 Each member of the NADPH oxidase family contains a catalytic unit termed Nox that forms a heterodimer with a lower molecular weight subunit called p22phox; this heterodimeric cytochrome is the site of electron transfer from NADPH to molecular oxygen, resulting in the formation of O2●−.36, 38 Cardiovascular cells exhibit specific patterns of Nox expression, with several cell types expressing more than one isoform. Nox1 is highly expressed in cultured vascular smooth muscle but it is not significantly expressed in cardiomyocytes or endothelial cells.39 Nox2 is abundantly expressed in cardiomyocytes, endothelial cells, and fibroblasts. Nox4 appears to be the most widely cardiac expressed isoform, being 9 ACS Paragon Plus Environment


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found in endothelial cells, cardiomyocytes and fibroblasts.36 The activity of nonphagocytic NADPH oxidase is significantly enhanced by several stimuli, namely angiotensin II, noradrenaline, tumor necrosis factor-α, endothelin-1, and cyclic stretch 38, 40-42

which act both through acute post-translational modification of oxidase,

regulatory subunits and by transcriptional pathways. NADPH oxidases have been shown to contribute to ROS-induced cardiac myocyte hypertrophy, contractile dysfunction, heart failure, and cardiomyocyte death.36 p66Shc can negatively regulate RAS-activation by means of displacing the nucleotide exchange factor Son of Sevenless (SOS) from its complex with GRb2. This chain of events can be accompanied by the SOS-dependent activation of the small GTPase Rac-1, which consequently promotes the assembly of membrane-bound NADPH-oxidases and the production of ROS.43 Moreover, p66Shc is a lifespan-regulating protein that contributes to mitochondrial ROS metabolism and to the regulation of the mitochondrial apoptosis pathway that catalyzes electron transfer from cytochrome c to oxygen.1, 44, 45 Thus, p66Shc contributes to mitochondrial H2O2 formation 44-46 and reduces the expression of antioxidant enzymes, namely glutathione peroxidase-1 (GPX-1) and MnSOD by downregulation of forkhead-type transcription factors (e.g. Foxo3a).45 Reactive nitrogen species (RNS) are also formed in high amounts in the heart. The most relevant RNS, ●NO, is formed from L-arginine by 3 isoforms of ●NO synthases (NOS): the constitutive isoforms, neuronal NOS (nNOS/NOS1) and endothelial NOS (eNOS/NOS3), and the cytokine-inducible NOS (iNOS/NOS2). The constitutive eNOS or NOS-III is not restricted to the endothelium, as it is also present in cardiac myocytes and may form small quantities of ●NO. eNOS is the predominant NOS isoform in the heart and is preferentially located in caveolae of the sarcolemma, in the vicinity of βadrenergic receptors and L-type calcium channels.47 nNOS has been found in the sarcoplasmic reticulum and is associated to the ryanodine receptor calcium release channel.47 In the healthy heart, the expression of iNOS is very low, however it can significantly increase during pathological conditions such as cardiac infarction or inflammation 1. Moreover, in the absence of sufficient amounts of substrate (L-arginine) or co-factors (tetrahydrobiopterin), NOS can produce O2●− 48 instead of ●NO. Furthermore, ●NO and O2●− combine spontaneously to form ONOO− that is a major source of potential damage to the heart.49 For simplicity, we will refer as oxidative stress to the overall damage caused by ROS and RNS.


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2. Drugs involved in toxicity towards the heart

2.1. Catecholamines

Catecholamines are the foremost and probably oldest studied molecules for their cardiac actions. They are well recognized regulators of cardiac function, but may also elicit cardiotoxic effects. Noradrenaline, adrenaline, and dopamine are biogenic catecholamines derived from the amino acid tyrosine 50 (Figure 1). Isoproterenol is usually used as a synthetic model for catecholamine toxicity since it has a biogenic amine-like structure and is a β-agonist. Catecholamine-induced necrosis and fibrosis in the myocardium of human patients was described as early as 1937 in a case of adrenaline misuse in asthma treatment.1 Also, noradrenaline has been reported to be fatal, namely after its prolonged infusion for treatment of shock, where focal myocarditis with degeneration of myofibrils and infiltration of leucocytes were observed.51 Pheochromocytoma patients show similar lesions in the heart, attributed to the high levels of circulating catecholamines.52 Furthermore, the levels of circulating catecholamines have been described to be high in septic shock.53 Several pathways are activated upon catecholamine stimulation that can lead to cardiac toxicity. One of these mechanisms is oxidative stress, although others can be equally or more relevant depending on the situation. Catecholamine-induced oxidative stress has been confirmed in both in vitro and in vivo models. Exposure to catecholamines resulted in increased lipid peroxidation, 54, 55 GSSG formation, 17, 56, 57 and the injury caused by catecholamine exposure was reversed or attenuated by antioxidants.54, 55, 58 Malondialdehyde-associated modification of proteins increased in catecholamine-perfused hearts.59 Dhalla and co-workers found that vitamin E prevented the transition from compensated hypertrophy to failure in guinea pigs with pressure-overload due to aortic constriction caused by catecholamines.60 Similarly, dimethylthiourea, a HO● scavenger, prevented chamber dilation and pump dysfunction in infarcted mouse heart.61 Neri and co-workers showed in the rat heart that the GSH/GSSG ratio significantly decreased and malondialdehyde levels increased after noradrenaline administration, showing an oxidative stress state of lipid peroxidation in the cardiac tissue, which was not reverted by propranolol (β-antagonist) or prazosin (α1-antagonist).62



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The sources of ROS formed upon catecholamines stimulation are most likely multifactorial, as they can result from adrenoceptor stimulation and/or from the enzymatic and non-enzymatic degradation of catecholamines.1 The activation of α1adrenoceptors, coupled with G-protein Gq, triggers NADPH oxidase activity in cardiomyocytes thus resulting in O2●- formation.38, 63 Prolonged noradrenaline treatment increases ROS production in fetal rat hearts and ventricular myocyte H9c2 cells via a selective increase in Nox1 expression.64 Noradrenaline-induced formation of ROS resulted in increased methylation of protein kinase Cε promoters which decreases protein kinase Cε gene expression. N-acetylcysteine, diphenyleneiodonium, and apocynin blocked noradrenaline-induced ROS production and the promoters’ methylation, therefore restoring protein kinase Cε mRNA and protein to control levels. Accordingly, noradrenaline-induced ROS production, promoter methylation, and protein kinase Cε gene repression were completely abrogated by knockdown of Nox1 in cardiomyocytes. 64 β-Adrenergic receptor-induced toxicity is also dependent upon mitochondrial ROS production. Exposure to the β-adrenergic agonist isoproterenol caused a concentrationdependent increase in the mitochondrial ROS in mouse cardiomyocytes, which was similar to the exposure to forskolin, which directly stimulates cAMP formation.59 This stimulatory effect is mediated via cAMP–protein kinase A-dependent and calciumindependent signaling that did not affect mitochondrial membrane potential. Other intracellular pathways can be involved in ROS formed by β-adrenergic stimulation: overexpression of type 5 adenylyl cyclase exacerbates the cardiomyopathy induced by chronic catecholamine stress by decreasing MnSOD expression, resulting in oxidative stress intolerance. Accordingly, susceptibility caused by overexpression of Type 5 adenylyl cyclase to cardiomyopathy was suppressed by overexpression of MnSOD. Type 5 adenylyl cyclase is one of the major adenylyl cyclase isoforms in heart and it appears to be involved in the transcriptional regulation of MnSOD via the Sirtuin 1/FoxO3a pathway.65 β-stimulation for 15 or 30 min leads to intracellular ROS formation and lipid peroxidation that were prevented by antioxidants.55, 66 Although these studies suggest only a direct participation of adrenoceptor activation on catecholamine-induced oxidative stress, other studies demonstrated that catecholamine cardiotoxicity may stem from their ability to undergo oxidative conversion to aminochromes with concomitant production of ROS. Corroborating this postulate, the formation of catecholamine oxidation products was not prevented by adrenoceptor antagonists in laboratory animal hearts.62 It is feasible that the early intracellular ROS formation 12 ACS Paragon Plus Environment

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caused by biogenic catecholamines is mainly due to β-stimulation, which soon is desensitized by receptor down regulation. Even so, the oxidation of catecholamines continues over time, which is counteracted by antioxidants but not by β-blockers, 55, 62 indicating a chemical mechanism. There is substantial evidence showing that catecholamines may be converted to unstable o-semiquinones that, after deprotonation and loss of a second electron, give rise to the corresponding o-quinones. These processes lead to O2●− formation.1 At physiological pH, the partial deprotonation of the catecholamines side chain amine group can lead to an irreversible 1,4-intramolecular cyclization, a reaction that occurs through nucleophile attack of the nitrogen atom linked to carbon 6 of the quinone ring. In the case of adrenaline, the cyclization forms the leucoadrenochrome, which is then further oxidized to adrenochrome.67, 68 The oxidation of catecholamines occurs very slowly at physiological pH, however it is increased considerably by enzymatic or metal catalysis 68-71 or in the presence of O2●−. 56, 72-74

The proportion of oxidation products depends upon the specific catecholamine

that is being metabolized and the general redox environment present. The oxidation products of catecholamines have been demonstrated to produce organelle alterations, intracellular calcium overload, coronary spasm, myocardial cell damage, depletion of high energy stores, and ventricular arrhythmias.1 In addition, oxyradicals, which are known to generate oxidative stress and produce cardiotoxic effects are formed during the oxidation of catecholamines.75 Fenton reaction or other pro-oxidant mechanisms can facilitate the formation of highly reactive catecholaminederived products. The protection of H9c2 cardiomyoblast cell line against adrenalineinduced toxicity using various iron chelators was recently demonstrated.76 The products of oxidized catecholamines were shown to form complexes with iron. These complexes had significant redox activity, which could be suppressed by salicylaldehyde isonicotinoyl hydrazine (iron-chelating agent). Also, in H9c2 cells, a higher cytotoxicity of oxidized catecholamines than that of the parent compounds was observed, apparently through the induction of caspase-independent cell death, whereas coincubation of cells with salicylaldehyde isonicotinoyl was able to significantly preserve the cells’ viability. A significant increase in intracellular ROS formation was observed after the incubation of cells with catecholamine oxidation products, which was also significantly reduced by salicylaldehyde isonicotinoyl hydrazine.77 In isolated cardiomyocytes, exposure to high concentrations of adrenaline demonstrated its redox ability, reflected by GSH depletion, with formation of GSSG, adrenaline-o-quinones, adrenochrome, and several other highly reactive species namely HO● and ONOO–.56, 78 This oxidative stress mediated by high levels of 13 ACS Paragon Plus Environment


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catecholamines was potentiated by the presence of other sources of ROS.17, 78 Moreover, the high chemical reactivity of catecholamine oxidation products makes them very hard to detect, especially in biological matrixes such as plasma or blood.71, 79 Many studies underestimate adrenaline-redox ability, while other experiments use supplemented medium with antioxidants to avoid catecholamine oxidation (i.e. ascorbic acid), since authors do not exclude the contribution of catecholamine oxidative metabolites to the observed effects.63, 80, 81 Furthermore, the pharmacological approaches taken in clinical situations where catecholamine levels are high, like heart failure or ischemia-reperfusion, seem to demonstrate that the oxidative stress mediated by catecholamines may not only depend on adrenoceptor stimulation. The ability of angiotensin converting-enzyme inhibitors or β-antagonists to suppress O2●– formation in vitro is also well-known.82-85 The oxidative stress observed after 8 weeks in a genetic model of sympathetic hyperactivity-induced heart failure (α2A/α2C-adrenoceptors knockout mice) is caused by direct catecholamine oxidation 86, as β-receptor blockage by metoprolol (does not have relevant antioxidant properties) did not prevent it, while carvedilol (third generation non-selective β-adrenoceptor antagonist with anti-α1adrenoceptors vasodilator effect and antioxidant properties) did. In fact, carvedilol led to increased GSH/GSSG ratio in carvedilol-treated heart failure mice.86 Additionally, captopril and zofenopril are sulfhydryl angiotensin converting-enzyme inhibitors able to scavenge HO● and decrease lipid peroxidation and phospholipid degradation during reperfusion injury where high levels of catecholamines are reached.87, 88 Additionally, SQ 14,534, the isomer of captopril, which is 100-fold less potent as an angiotensin converting-enzyme inhibitor but equipotent in scavenging radicals, also improves reperfusion-induced cardiac dysfunction.89 In vitro, the oxidation of adrenaline to adrenochrome mediated by O2●− is inhibited by captopril, SQ 14,534, and zofenopril but not by angiotensin converting-enzyme inhibitors with no relevant antioxidant proprieties.89 The antioxidant aspects of these clinical useful compounds can in fact contribute to better clinical outcomes in diseases where increased catecholamines levels represent poor prognostic. These molecules easily reach important pharmacological concentrations in the heart, where most antioxidant therapies may not. Besides, chronic conventional antioxidant therapy has shown pro-oxidant effects.1 In addition to adrenergic receptor-dependent mechanisms and non-enzymatic oxidation of the molecules, enhanced MAO activity coupled with increased intramyocardial catecholamines availability results in augmented ROS generation. Altogether, they contribute to maladaptive remodeling and left ventricular dysfunction in hearts subjected to chronic stress.90 In fact, MAO inhibitors have been found to decrease the


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incidence and severity of myocardial lesions following catecholamine administration.91, 92

Several cardiovascular effects of centrally acting and medically useful drugs have been reported for the past years (e.g. tricyclic antidepressants, antipsychotics, anticonvulsants, or barbiturates). Most of those molecules interfere with cardiac ion fluxes or interact with central and peripheral neurotransmitter systems (mainly catecholaminergic) with noxious cardiac consequences.93 Numerous other factors may contribute to explain the cardiac effects of these drugs, 93 but were considered out of the scope of this review since they are not oxidative stress related. The toxicity of catecholamines and their ability to generate oxidative stress are unquestionable in spite of the multiple mechanisms proposed and the still ongoing discussion regarding the most relevant mechanism.1, 54, 94-96 For a wide comprehensive review regarding the contribution of the oxidation products of catecholamines and heart pathology see Costa et al. 2011.1

2.2. Drugs of abuse

Amphetamines and cocaine (Figure 1) are cardiotoxicants, as reported by human studies and case reports. The most obvious mechanism for their cardiac toxicity is related to their ability to activate the catecholaminergic system in the central nervous system (CNS) and peripheral organs. This catecholaminergic activation leads to the direct lesion of the heart or coronary system as referred in the last section reporting to catecholamines. However, several studies have proven that the direct action of amphetamines and/or their metabolites can trigger cardiotoxicity unrelated to catecholamine released (for a wide review of amphetamines toxicity to target organs please see Carvalho et al. 2012). 97

2.2.1. Methamphetamine and amphetamine

Methamphetamine is easily available on the streets and most of its clinical complications are related to heart or CNS injuries. Mechanistically, amphetamines 15 ACS Paragon Plus Environment


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mainly act on the CNS causing the release of monoamine neurotransmitters, including dopamine, noradrenaline, and serotonin.98 In fact, autopsies of deaths attributed to methamphetamine use have shown contraction band necrosis in the myocardium that are clinical signs of catecholamine toxicity, thus corroborating the involvement of biogenic amines on its cardiotoxicity.99, 100 Methamphetamine is well known to elicit rapid tachycardia in humans that persists for several hours 101, 102 with increased systolic and diastolic blood pressures. Cerebral stroke, hemorrhage, and arrhythmias have also been reported to be associated with methamphetamine abuse.103, 104 Postmortem studies on cardiac tissue show that methamphetamine causes myocardial infarction, cardiomyopathy, and ventricular hypertrophy.100, 105-109 Methamphetamine-elicited oxidative damage in the heart has been studied in animal models but data are still scarce. In rats subjected to four methamphetamine binges [3 mg/kg, intravenous (i.v.) for 4 days, separated by a 10-day drug-free period], cardiac oxidative stress was detected.110 Dihydroethedium staining showed that methamphetamine significantly increased the levels of ROS in the left ventricle. Treatment with the SOD mimetic, tempol (2.5 mM), in the drinking water prevented methamphetamine-induced left ventricular dilation and systolic dysfunction. Also, tempol significantly reduced, but did not eliminate dihydroethedium staining in the left ventricle. Moreover, tempol did not prevent the diastolic dysfunction.110 Methamphetamine-induced oxidative damage might be related to the high levels of catecholamines as the result of its interaction with neurotransmitter transporters and the catecholamines enzymatic degradation system.97 Another important factor that may contribute to methamphetamine-induced oxidative stress in the heart is inflammation. 99

The heart of rats treated with a methamphetamine binge regimen showed focal

inflammatory infiltrates with abundant monocytes and occasional necrotic foci.111 Inflammatory processes per se lead to the formation of ROS during the ‘oxidative burst’.

112

In particular, mitochondrial dysfunction, the oxidative and nitrosative stress

(evaluated by tyrosine nitration) 110 and inflammation 99, 111 caused by the methamphetamine-induced catecholamine surge are crucial for the impairment of cardiac function and it may extend well beyond the acute pharmacodynamic effects of methamphetamine, representing an underlying and potentially progressive degenerative process. Amphetamine is a drug of abuse whose structure and mechanisms are similar to methamphetamine, although the former has a shorter half-life. It is a sympathomimetic agent that stimulates catecholamine release, particularly dopamine and noradrenaline, from the presynaptic nerve terminals.97 Amphetamine is also used as a therapeutic 16 ACS Paragon Plus Environment

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drug, namely as mixed amphetamine salts, or as the prodrug, lisdexamfetamine.113-115 Despite the well-established efficacy and tolerability profile of amphetamine derivatives in attention-deficit hyperactivity disorder therapy 115, there is concern about the potential for rare but serious cardiovascular adverse events, namely sudden cardiac death.113, 114 Cardiotoxicity (manifested as cardiomyopathy, acute myocardial infarction/necrosis, heart failure, or arrhythmia) after the use or misuse of amphetamine has been widely documented.97 Amphetamine causes myocardial infarction and coronary spasm among other cardiovascular effects.116-119 Despite case reports of cardiac complications resulting from amphetamine use, those effects have been linked with the catecholaminergic effects of amphetamine and other derivatives, since few mechanistic studies have been performed. In rats, after 14 days of daily d-amphetamine administration, heart cysteine levels were significantly depleted when compared to the pair-fed group, although the two groups had similar heart antioxidant enzyme levels (CuZnSOD, MnSOD, catalase, GPX, GR and GST).120 The single administration of d-amphetamine (20 mg/kg) stimulated the production of H2O2 in CD-1 mice heart, in a MAO-dependent manner, 121 thus demonstrating the importance of the oxidative stress-induced by catecholamine release. These data demonstrate the link between amphetamine and cardiac oxidative stress. However, despite numerous human case reports, more mechanistic studies should be warranted in the future to fully disclose whether its actions are only related to catecholaminergic system activation or if other pathways are involved.

2.2.2. 3,4-Methylenedioxymethamphetamine (MDMA, ‘ecstasy’)

In humans, the oral administration of 3,4-methylenedioxymethamphetamine (MDMA) (doses ranging between 75 to 175 mg) produces acute sympathomimetic effects, such as increased heart rate and blood pressure, and transient anxiety.122-125 MDMA related fatal complications have been associated to cardiac abnormalities, like arrhythmias, 126 massive neurological disturbances, and multiple organ failure.127-131 MDMA, like other amphetamines, increases the release of peripheral and central monoamines. MDMA causes the efflux of serotonin and noradrenaline and to a lesser extent dopamine by an exchange diffusion process involving the respective transmitter transport carriers.132 In vivo experimental studies show that MDMA administration leads to oxidative stress. After administration of 20 mg/kg intraperitoneal (i.p.) of MDMA, the activities of 17 ACS Paragon Plus Environment


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antioxidant enzymes, namely GPX, SOD, and GR in the heart were significantly reduced, even just a few hours (3 or 6 h) after treatment.133 The levels of vitamin C and GSH decreased significantly while malondialdehyde levels increased after MDMA treatment.133 Shenouda et al. observed an increase in nitrated tyrosine residues, which is as a marker of oxidative stress.134 As previously stated, ONOO

is formed by a

diffusion-limited reaction of both superoxide anion radical (O2 ) and ●NO 1 and ONOO ●_

leads to the nitration of tyrosine residues of several proteins. In rats exposed to MDMA [9 mg/kg i.v. twice daily (morning and afternoon) for 4 days and followed by a 10 day drug-free period], proteomic analysis revealed increased nitration of contractile proteins (troponin-T, tropomyosin alpha-1 chain, myosin light polypeptide, and myosin regulatory light chain), mitochondrial proteins [Ub-cytochrome-c reductase and adenosine triphosphate (ATP) synthase], and sarcoplasmic reticulum calcium ATPase, 134

revealing a high nitrosative stress caused by the drug.

In contrast, in vitro studies do not show any significant link between MDMA and oxidative stress: no direct increase of reactive species occurs after MDMA incubation nor alterations in intracellular antioxidants after exposing cardiac cells to MDMA. 135 In vivo, MDMA is metabolized into redox active metabolites, namely alpha-methyl dopamine, N-methyl alpha-methyl dopamine, and their correspondent glutathione metabolites.132 The MDMA metabolites, especially the reactive (and unstable) orthoquinones, trigger the formation of large quantities of reactive oxygen and nitrogen species.97, 136 Actually, alpha-methyl dopamine, N-methyl alpha-methyl dopamine, and 2,5-bis(glutathion-S-yl)-alpha-methyl dopamine cause oxidative stress and contractile dysfunction in adult rat left ventricular myocytes. All three metabolites of MDMA induce time- and concentration-dependent increases in ROS that were prevented by N-acetylcysteine.136 Carvalho et al. demonstrated that incubation of isolated adult rat cardiomyocytes with MDMA metabolites, N-methyl-α-methyldopamine and αmethyldopamine, resulted in loss of normal cell morphology and a decrease in GSH levels and antioxidant enzyme activities.137 Therefore, in vivo cardiac MDMA-induced oxidative stress can be due, at least in part, to the metabolism of MDMA to its redox active metabolites. It is a known fact that MDMA-induced oxidative stress plays an important role in its cardiotoxic effects, as confirmed by the animal studies and human reports. Its cardiotoxicity is often related to MDMA actions upon neurotransmitters release and reuptake. It is however feasible that the redox active metabolites of MDMA can also play an important role in heart injury by promoting increased oxidative stress.


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Therefore, the metabolism of this drug should not be neglected when evaluating its cardiotoxic actions.137, 138

2.2.3. Cocaine

Cocaine is an extremely powerful psycho-stimulant with highly addictive properties.139 Cocaine overdose is the most frequent cause of drug of abuse related death reported by medical examiners in the United States, and those fatal events are most often related to the cardiovascular manifestations of the drug (Figure 2). There is a wellestablished link between cocaine use and myocardial infarction, arrhythmia, heart failure, and sudden cardiac death.139, 140 Several mechanisms have been postulated to explain how cocaine contributes to these conditions, but the cardiovascular complications related to cocaine abuse are mainly adrenergically-mediated.139 Cocaine is a stimulant of the sympathetic nervous system through multiple actions: inhibition of catecholamine reuptake, stimulation of central sympathetic outflow, and by increasing the sensitivity of adrenergic nerve endings to noradrenaline.141 The increase in adrenergic activity, along with the local anesthetic effect caused by sodium channel inhibition, results in increased myocardial contractility, heart rate (up to 34%), systemic arterial pressure (up to 15%), and myocardial oxygen demand. The transient flux of Ltype calcium channels across the cell membrane may also be altered (increased) by cocaine.140 In summary, the arrythmogenic potential of cocaine can be mediated by the blockage of potassium channels, increase of L-type calcium channel current, and inhibition of sodium influx during depolarization.141 Additionally, long-term cocaine use has been associated with left ventricular hypertrophy, myocarditis, and dilated cardiomyopathy, favoring heart failure if drug use is continued (Figure 2). 142, 143 As in amphetamines, the stimulant effects of cocaine are mediated by changes in synaptic concentrations of biogenic monoamines; however, cocaine alters monoamine levels through different mechanisms. Cocaine is a CNS stimulant affecting the release and reuptake of serotonin and catecholamines in the brain. It blocks the reuptake of noradrenaline and dopamine at peripheral preganglionic synaptic nerve endings, thus increasing the synaptic concentrations of these monoamines. Cocaine also causes the release of noradrenaline and adrenaline from the adrenal medulla. Thus, cocaine is a powerful sympathomimetic agent that can cause significant central and peripheral vasoconstriction.140 Cocaine has a rapid and high uptake and rapid clearance from the 19 ACS Paragon Plus Environment


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heart.144 Though the overall stay of cocaine in the heart is short, prolonged inhibition of the noradrenaline transporter has been shown (as assessed by 6-18Ffluoronoradrenaline), demonstrating that the effects of cocaine on cardiac neurotransmitter activity can persist long after the drug has been cleared. 139 Furthermore, cocaine administration rapidly increases vesicular transport by vesicular monoamine transporter-2 (VMAT-2).145 The adrenergic overstimulation induced by cocaine is highly correlated with its ability to increase oxidative stress. Repeated i.p. cocaine administrations in vivo led to significant increases in cardiac malondialdehyde, GSSG, and protein carbonyls, while levels of GSH, MnSOD, GPX, catalase, and GST decreased.142 Moreover, peroxidation of heart membrane phospholipids was observed in rats during the maintenance of selfadministration of cocaine.146 The ability of cocaine to cause oxidative stress was further corroborated by a study of Fineschi and co-workers.147 After chronic cocaine administration to rats for 30 days, GSH was significantly depleted in the heart at 30 min to 24 h after administration, and GSSG increased in a similar time frame. Moreover, in the same study, SOD increased during the first hour, GR and GPX both increased between 30 min and 24 h, possibly as an adaptive response to the increased oxidative stress caused by cocaine. The ascorbic acid levels increased after 1 h and remained significantly so for 24 h, while malondialdehyde increased between 30 min and 24 h. In this study, noradrenaline was administrated as well, in a similar setting to that of the chronic cocaine administration. In this experimental model, cocaine administration was associated with a significant increase in heart weight/body weight ratio that was comparable to noradrenaline-induced cardiac injury, linking the cardiac toxicity of cocaine to its noradrenergic stimulating effect.147 In fact, when comparing the cocaine and noradrenaline experiments, the frequency and extent of cardiac lesions obtained with 40 mg/kg × 10 days + 60 mg/kg × 20 days of cocaine were similar to those with 8 mg/kg of noradrenaline single administration and sacrifice 24 h after.147 In another study, after 2 days of cocaine administration to rats (total of 3 administrations of 7.5 mg/kg/day, i.p.) no differences were observed in echocardiographic parameters between the cocaine-treated and the control group.148 However, an increase in oxidative stress in the myocardium was demonstrated through increases in lipid peroxidation, a huge increase in antioxidant enzymes and in NADPHdriven O2●_ production. Furthermore, higher gp91phox and p22phox mRNA expression, measured by quantitative real-time RT-PCR, was found in the cocaine group. Another protocol of the same study, cocaine hydrochloride (2x7.5 mg/kg/day, i.p.) was administered for 7 days; 24h after the last administration, the evaluations were 20 ACS Paragon Plus Environment

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made.148 Cocaine administration induced a cardiac dysfunction, characterized by a decrease in cardiac index and left ventricular fractional shortening. This left ventricular dysfunction was prevented by antioxidant treatment (100 mg/kg/day vitamin C and 100 U/kg/day vitamin E). Moreover, in these animals, the antioxidant treatment decreased lipid peroxides and decreased the activity of NADPH oxidase, linked to the down regulation of gp91phox. These data show that cocaine administration induces early NADPH-driven O2●_ release, which may play an important role in the development and progression of left ventricular dysfunction observed in chronic cocaine abuse.148 Some of these data were confirmed in a mouse model of chronic escalating binge cocaine administration: days 1 to 4 at 3x15 mg/kg, days 5 to 8 at 3x20 mg/kg, days 9 to 12 at 3x25 mg/kg, and days 13 to 14 at 3x30 mg/kg.149 Compared to controls, chronic binge cocaine administration significantly increased the cardiac NADPH-dependent O2●_ production. The cocaine-induced ROS production was associated with significant increases (approximately 2-fold) in protein expression of Nox2 (an isoform of NADPH oxidase) and its regulatory subunits: p22phox, p67phox, p47phox, p40phox, and Rac1; and in p47phox phosphorylation. Increased Nox2 activity was accompanied by the activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase in cardiomyocytes.149 Moreover, dysfunctional NOS activity can also contribute to ROS production in cocaine-treated groups. 149 This work by Fan and collaborators was complemented by an in vitro study with H9c2 cells, where cocaine-induced ROS production was primarily a direct action of cocaine on cardiac myocytes that caused severe oxidative damage to myocytes and cell death in concentrations (at µM) reported by the authors as easily obtained in cocaine-consuming humans. Also, cocaine-induced ROS formation was reverted by inhibitors of protein kinase C (bisindolymaleimide) or by depletion of Nox2 using small interfering RNA, 149 therefore demonstrated the cocaine oxidative stress potential in the absence of catecholamines. Without doubt, cocaine increases oxidative stress in the heart although doubts still persist concerning all the factors involved. Cocaine is able per se to cause oxidative stress, while in in vivo settings, adrenergic signals can further escalate the severity of cocaine-related oxidative stress and the toxicity towards the heart. Neutrophils infiltration and activation of NADPH oxidases and NOS strongly contribute to oxidative stress induced by cocaine. The cocaine metabolites have been linked to toxicity in kidney and liver 150, however at this point very little is known about their potential toxicity to the heart. A study by McCance and collaborators determined however that the cardiovascular effects (increased heart rate and systolic blood pressure) of cocaethylene and cocaine were similar in humans.151 21 ACS Paragon Plus Environment


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2.3. Ethanol

Alcoholism is one of the major causes of non-ischemic heart damage. Chronic alcoholism has been demonstrated to cause detrimental damage to cardiac muscle with the development of alcoholic cardiomyopathy which accounts for ~33% of all dilated cardiomyopathies. In patients with chronic alcoholism, the occurrence of cardiomyopathy of any kind is increased by 50%, with a majority of these patients dying from heart failure. Alcoholic cardiomyopathy is manifested as cardiomegaly, disrupted myofibrillar architecture, reduced myocardial contractility, decreased ejection volume and enhanced risk of stroke and hypertension. Several mechanisms have been postulated for alcoholic cardiomyopathy including oxidative damage, accumulation of triglycerides, altered fatty acid extraction, decreased myofilament calcium sensitivity, and impaired protein synthesis.152 Ethanol’s ability to cause cardiac oxidative stress has been widely proved. In vitro studies were performed in cardiomyocytes isolated from 1- to 2-day-old SpragueDawley rats that were incubated with 50, 100, or 200 mM of ethanol for 24 h. The cells showed typical apoptosis in the lower doses tested (up to 100 mM), while the number of necrotic cells greatly increased in 200 mM ethanol-treated cells.153 Intracellular production of reactive oxygen intermediates increased, while mitochondrial membrane potential decreased after ethanol treatment. Both vitamin E (1 mM) and vitamin C (0.2 mM) inhibited the oxidative stress and the myocyte apoptosis observed.153 The underlying mechanism of cardiomyocyte death appears to involve mitochondrial damage through an increase in oxidative stress whereas antioxidants, to a large extent, inhibit oxidative stress and apoptosis induced by ethanol.153, 154 In vivo studies performed in animal models also demonstrated oxidative stress involvement in cardiac toxicity of ethanol. Adult female Friend Virius B-type mice were treated with ethanol by oral gavage at a dose of 5 g/kg.155 Six hours after treatment, ethanol-induced myocardial injury was observed as indicated by a significant increase in serum creatine phosphokinase activity and myocardial ultrastructure alterations, predominantly mitochondrial swelling with cristae disarray. The myocardial injury was linked to a significant increase in myocardial lipid peroxidation and protein oxidation. Acute alcohol exposure decreased GSH content in the heart, more in the mitochondria than in cytosol. These alcohol-induced myocardial injuries and changes in 22 ACS Paragon Plus Environment

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oxidative stress parameters were all significantly reverted by supplementation with Nacetyl-L-cysteine prior to alcohol exposure.155 Furthermore, in another study, the cardiac-specific overexpression of catalase in a transgenic mouse line rescued ventricular myocytes from ethanol-induced cardiac contractile defects.156 After 35 days of drinking a 30% ethanol-solution, male Wistar rats had higher heart weight/body weight ratio and exhibited increases in myocardial lipid hydroperoxides, indicating alcoholic heart disease with hypertrophy and oxidative stress.157 Myocardial SOD activity was higher, whereas GPX activity was lower in ethanol treated rats when compared to control. The co-administration to 2g/L N-acetyl-L-cysteine led to lower myocardial lipid hydroperoxide and higher GPX activity compared to animals just drinking ethanol, thus demonstrating oxidative stress involvement in ethanol cardiotoxicity.157 The overexpression of alcohol dehydrogenase (ADH) (enzyme that oxidizes ethanol into acetaldehyde) exacerbates the ethanol-induced cardiac depression. The major ethanol metabolite acetaldehyde has allegedly a causative role in the onset of the myopathy state because of its ability to accumulate in the heart and its highly reactive properties when compared to ethanol.158 In fact, 33% patients with alcoholic heart muscle disease had antibodies against cyanoborohydride-stabilized acetaldehyde-modified human cardiac cytosolic protein antigens.159 The generation of transgenic mice that overexpress ADH and the acetaldehyde-metabolizing enzyme mitochondrial aldehyde dehydrogenase 2 (ALDH2) helped to elucidate the role of acetaldehyde in alcohol-induced toxicity. 160 Following 8-12 week feeding with 4% alcoholic diet, mechanical function was depressed in wild-type cardiomyocytes and was characterized by reduced peak shortening, impaired myocyte re-lengthening, and dampened intracellular calcium release and sarcoplasmic reticulum calcium reuptake.160 Enhanced oxidative stress, lipid peroxidation, and protein carbonyl formation were observed in alcohol consuming mice. Moreover, ADH overexpression exaggerated, whereas ALDH2 overexpression attenuated, alcohol-induced mechanical and intracellular calcium defects, oxidative stress, lipid peroxidation, and protein damage, 160 thus demonstrating the toxic effects of acetaldehyde. Other works have demonstrated the potential cardiotoxicity of the highly reactive metabolite, mainly linking its effects with mitochondrial damage.161 As stated, mitochondria dysfunction is responsible for cardiac oxidative stress. Both ethanol and acetaldehyde have been found to alter energy-forming mechanisms in the heart 162, 163, while others studies do not report it.164



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The models and concentrations used are the main differences among those studies. Transgenic models where increase cardiac levels of acetaldehyde are found give strong arguments in favor to its role on cardiotoxicity. In in vitro settings using human cardiac myocytes, acetaldehyde and ethanol increase ROS formation and apoptosis following 24-48 h of incubation. 165 However, the levels of ROS formed after 24 h with 100 µM acetaldehyde were similar to the levels formed by 100 mM of ethanol, under the same experimental conditions. The transfection of human cardiac myocytes to promote ALDH2 overexpression and evaluate acetaldehyde- and ethanol-induced cell injury was also used.165 As expected, the human ALDH2 transgene significantly attenuated acetaldehyde-induced and ethanol-induced ROS generation and apoptosis. In addition, the promotion by acetaldehyde and ethanol of ROS formation and of apoptosis was antagonized by non-enzymatic antioxidants, namely N-acetyl-L-cysteine and vitamin E. The human ALDH2 overexpression-elicited protection against ethanolinduced ROS formation was ablated by the ALDH inhibitor, cyanamide.165 The data gathered in ethanol-induced cardiomyopathy is undoubtedly linked to oxidative stress through several mechanisms. The acetaldehyde reactivity and mitochondrial dysfunction seem to be the most important contributors to ethanol cardiac oxidative stress. Acetaldehyde accumulates easily in the heart 161 and has a high reactivity towards cellular nucleophiles, 166 while ethanol (and possibly also acetaldehyde) causes mitochondrial dysfunction.154 This dual response (acetaldehyde reactivity towards cellular antioxidant nucleophiles and ethanol-induced mitochondrial damage) are the main contributors to oxidative stress in alcoholic cardiomyopathy.

2.4. Anticancer drugs

Cancer is increasing worldwide. In Europe alone an estimated 3.45 million new cases of cancer (excluding non-melanoma skin cancer) and 1.75 million deaths from cancer occurred in 2012.167 Furthermore, anticancer treatments can induce severe damage to non-cancer tissues, which challenge patients’ compliance and quality of life 168, 169. The cardiac tissue is very susceptible to the side effects of several chemotherapeutic agents.168-170 Cardiotoxicity in anticancer therapy has been described for almost 30 years. Anthracycline and anthracenedione cytostatic antibiotics are the best known chemotherapeutic agents that cause cardiotoxicity. Alkylating agents such as cyclophosphamide, ifosfamide, cisplatin, carmustine, busulfan, chlormethine, and 24 ACS Paragon Plus Environment

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mitomycin have also been associated with cardiotoxicity. Other agents have also been linked to cardiac dysfunctions namely paclitaxel, etoposide, teniposide, the vinca alkaloids, fluorouracil, cytarabine, amsacrine, cladribine, asparaginase, tretinoin, pentostatin, human epidermal growth factor receptor 2 (HER2) and tyrosine kinase inhibitors. Cardiotoxicity is less frequent in some of the previously mentioned agents, but it may occur in up to 20% of patients when they are subjected to combined therapy with doxorubicin, daunorubicin, cyclophosphamide, mitoxantrone or fluorouracil 168, 169, 171

(Table 1).

Cardiac events related to chemotherapeutic therapy may include mild blood pressure changes, thrombosis, electrocardiographic changes, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, reduced left ventricular ejection fraction (LVEF), and congestive heart failure. These may occur during or shortly after treatment, within days or weeks after treatment, or may not be clinically relevant until months, and sometimes years, after the end of chemotherapy cycles.171 Several risk factors may predispose a patient to cardiotoxicity, namely cumulative total dose (anthracyclines, mitomycin, anthracenediones); total dose administered during a day or a course (cyclophosphamide, ifosfamide, carmustine, fluorouracil, cytarabine); rate of administration (anthracyclines, fluorouracil); schedule of administration (anthracyclines); mediastinal radiation; age; female gender; concurrent administration of cardiotoxic agents; prior anthracycline chemotherapy; history of pre-existing cardiovascular disorders; electrolyte imbalances such as hypokalemia and hypomagnesaemia; and in some particular cases (e.g. fluorouracil), genetic factors can also contribute to cardiotoxicity.171 The subclinical decline in cardiac function occurs most likely than congestive heart failure in result of anticancer therapy. Actually, the observed differences in results between clinical trials and some postmarketing studies are likely due to the variation in total follow-up, patient populations, timing of laboratory measurements, monitoring techniques (such as MUGA vs echocardiogram), and criteria for cardiotoxicity determination. Notably, even the definition of relevant decreased LVEF has been found inconsistent among studies.172, 173 Anthracyclines and anthracenediones are important oncotherapeutics with established efficacy in a variety of tumor types; however, their use is associated with irreversible and cumulative cardiotoxicity and therefore will be deeply addressed in this review. Cyclophosphamide and paclitaxel are referred as being responsible for cardiac toxicity. The therapy for cancer usually encloses a cocktail of cytostatic drugs, some of them acknowledged as cardiotoxic, therefore a section will address the multiple



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administration of anticancer drugs and the synergistic potential of the combination for promoting cardiac damage through oxidative stress mechanisms (Table 1).

2.4.1. Doxorubicin and other anthracyclines

Anthracycline-induced cardiotoxicity is a progressive disease that evolves in a stepwise fashion, with dramatic changes in cardiac ultrastructure that ensue before any clinical manifestations of cardiac dysfunction occur.174 Depending on the dose, pharmacokinetics and type of anthracycline (doxorubicin, epirubicin, daunorubicin or idarubicin) used, myocardial cell loss and/or functional impairment may occur. Initially, a relatively modest decline in contractile function usually is observed because intrinsic and extrinsic physiologic cardiac mechanisms are able to compensate for myocardial cell loss and decline in pump function. However, after additional cardiac insult and secondary damage, left ventricular remodeling and symptomatic heart failure may develop after anthracycline therapy.173 Despite of their dose-dependent cardiotoxic effects, anthracyclines remain widely used because of their high efficacy in the treatment of several tumors. In particular, they are valuable in breast and esophageal carcinomas, osteosarcoma, Kaposi sarcoma, softtissue sarcomas, Hodgkin’s disease, and Non-Hodgkin's Lymphoma.168 Doxorubicin use is limited by the risk of developing cardiomyopathy and congestive heart failure, and the probability to those events hugely increases when patients receive a cumulative dose above 500–550 mg/m2.175 One of the first retrospective analyses on doxorubicin therapy examined 4018 patient records and observed an overall incidence of drug-induced congestive heart failure of 2.2% (88 cases).175 The overall incidence of cardiotoxicity was later observed to be higher and comprised multifactorial aspects not first considered. In a study with 630 patients, 32 had a diagnosis of congestive heart failure after anthracycline use.176 Estimates indicate that about 26% of patients would experience doxorubicin-related congestive heart failure at a cumulative dose of 550 mg/m2. Age appeared to be an important risk factor for doxorubicin-related congestive heart failure after a cumulative dose of 400 mg/m2, with older patients (age > 65 years) having a higher incidence of heart failure compared to younger patients (age < or = 65 years). In addition, more than 50% of the patients who experienced doxorubicin-related congestive heart failure had a reduction (< 30%) in LVEF while the evaluations were ongoing.176


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All anthracycline analogs show cardiac toxicity and similar outcomes but with different cumulative doses. For each of the anthracycline analogs, the maximum tolerated cumulative dose will depend on the drugs’ established pharmacokinetic determinants; for example, the maximum tolerated dose for epirubicin is approximated to ∼900 mg/m2, primarily (but not exclusively) because it has higher glucuronidation and body clearance when compared to doxorubicin.169 Therefore, to limit cardiotoxicity, patients receiving anthracyclines are subjected to a maximum cumulative lifetime dose, which may result in premature discontinuation of the therapy and limits the use of anthracyclines following cancer relapse. Experimental work has clearly demonstrated the connection between anthracyclines and oxidative stress. Doxorubicin forms ROS in H9c2 cardiomyocytes, as shown by dichlorodihydrofluorescein oxidation and by the expression of stress-responsive genes that code for catalase or aldose reductase.177 Doxorubicin also increases ferritin levels in these cells, particularly the H subunit.177 It has been found that doxorubicin more selectively induces oxidative damage to the heart when compared to other organs. Lipid peroxidation is a major cardiotoxic indicator for doxorubicin, particularly under conditions of acute exposure. Kang et al. determined lipid peroxide levels in the heart caused by doxorubicin as a function of time (days) after administration (20 mg/kg). Lipid peroxide levels in the heart reached a peak value on the 4th day after the drug treatment 178. At clinically relevant doses, doxorubicin reduced cardiomyocytes contractility, sarcoplasmic reticulum calcium-ATPase (SERCA) protein and sarcoplasmic reticulum calcium-content, and glutathione reductase gene expression, while increasing oxidative stress.179 Furthermore, mice (7 weeks old) from several catalase overexpressing transgenic lines and from non-transgenic controls were treated with doxorubicin at a single dose of 20 mg/kg (i.p.) and sacrificed on the 4th day after the administration. Transgenic overexpressing catalase mice (60- or 100-fold higher activity) exhibited a significantly higher resistance to doxorubicin-induced cardiac lipid peroxidation, elevation of serum creatine phosphokinase, and to functional changes in the isolated atrium when compared to their non-transgenic paired controls. 178

The free radical hypothesis for doxorubicin acute cardiotoxicity was further

corroborated by reports showing that doxorubicin increases lipid peroxidation and oxygen species production in the heart tissue and plasma.180-182 Antioxidants like Nacetylcysteine 181, vitamin E, SOD 183, probucol 184, 185, and catalase 178 decrease the severity of acutely doxorubicin-induced oxidative stress.



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The current thinking is that anthracyclines may become cardiotoxic after one- or twoelectron reductive activation.169 Doxorubicin (and other related molecules) undergoes one-electron reduction through a metabolic activation caused by NADPHoxidoreductases.186 This futile cycle is supported by a number of NADPHoxidoreductases: cytochrome P450 or -b5 reductases, mitochondrial nicotinamide adenosine dinucleotide (NADH) dehydrogenase, xanthine dehydrogenase, endothelial nitric oxide synthase (reductase domain).187 The reduction by oxidoreductases generates a doxorubicin semiquinone. This semiquinone in the presence of molecular oxygen rapidly reduces it to O2●_, while the anthracycline is being regenerated back to its original quinone form. Superoxide is converted to H2O2 spontaneously or through the action of SOD. Moreover, the doxorubicin semiquinone can react with H2O2 to yield HO●.169, 188 This highly toxic ROS reacts with cellular molecules including nucleic acids, proteins, and lipids, thereby causing cell damage (Figure 3). Anthracyclines accumulate in the heart at concentrations 10- to 500-times higher than their extracellular concentrations; furthermore, in the intracellular medium, anthracyclines show a high affinity for cardiolipin, a lipid in the inner mitochondrial membrane. Therefore, the high intra-mitochondrial concentration of anthracyclines favors oxidative stress in that organelle, which impairs energy metabolism, alters mitochondrial DNA, and further enhances oxidative stress (Figure 3). 189, 190 During the redox cycle, the anthracycline semiquinone can also oxidize, losing the sugar moiety.191 This oxidation leads to the formation of more lipophilic compounds, the anthracycline aglycones, that easily intercalate in the mitochondrial membranes causing mitochondrial dysfunction (Figure 3).187 Due to their increased lipid solubility, aglycones intercalate in biologic membranes and form ROS in the closest vicinity to sensitive targets. Heart mitochondria seem to be such sensitive organelles, since aglycones also modify mitochondrial sulfhydryl groups 192 and induce a Ca2+independent oxidation of mitochondrial NADPH resulting in the production of O2•-.193 In 1997, using ESR techniques to analyze the identity and possible interactions of radical species emerging from NADH-respiring heart mitochondria in the presence of doxorubicin, Gille and Nohr showed that after one-electron transfer to the parent hydrophilic anthraquinone molecule, destabilization of the formed radical caused a cleavage of the sugar residue, with aglycone metabolite formation.194 Accumulation of the lipophilic aglycone metabolite in the inner mitochondrial membrane diverts electrons from the regular pathway to other electron acceptors out of sequence such as H2O2. HO

•

in that study was formed and affected the functional integrity of energy-

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Two electron reduction of the side chain carbonyl moiety is also a possible route of metabolization and results in the conversion of anthracyclines into secondary alcohol metabolites (such as doxorubicinol, epirubicinol). It is mediated by cytoplasmic cardiac aldo/keto or carbonyl reductases (Figure 3). 195 The alcohol metabolites are slightly less prone to redox cycling but remarkably potent at deregulating ion pathways, 196 namely calcium 197 and iron. The secondary alcohol metabolite doxorubicinol is able to release iron from its clusters and to inactivate cytoplasmic aconitase, an important posttranscriptional regulator of iron homeostasis. 198 The increased available ‘free’ iron largely contributes to the Fenton reaction and therefore amplifies oxidative stress.187 The increased polarity of doxorubicinol reduces its cardiac elimination when compared to doxorubicin, causing an even higher accumulation of total anthracyclines inside the cardiomyocytes. This became evident in studies of transgenic mice bearing cardiacrestricted overexpression of human carbonyl reductase, an enzyme that metabolizes doxorubicin to doxorubicinol. The human carbonyl reductase expressing hearts of transgenic animals produced four times more doxorubicinol. These animals also presented higher cardiac levels of total anthracyclines.199 Electron microscopy data showed swelling and major structural damage in the mitochondria of the human carbonyl reductase expressers, which led to the development of both acute and chronic cardiotoxicity.199 Acute cardiotoxicity was evident by a 60% increase in serum creatine kinase activity and a 5-fold increase in cardiac damage measured by electron microscopy. Furthermore, electrocardiograph telemetry and survival data were monitored during chronic doxorubicin-induced cardiotoxicity. The transgenic mice bearing cardiac-restricted overexpression of human carbonyl reductase developed cardiotoxicity 6-7 weeks before the non-expressers. The transgenic over-expressers survived for 5 weeks when compared to the 12 weeks of the non-trangenic animals with the same regimen of doxorubicin.199 These data allowed to raise the possibility that the cardiotoxicity of doxorubicin was enhanced not only by a facilitated formation and action of doxorubicinol, but also by a reduced elimination of anthracyclines from the heart, which may help to explain how anthracyclines present a lifelong risk for cardiotoxicity.169 The role of the redox active metal, iron, on doxorubicin-induced cardiotoxicity and its involvement in doxorubicin redox cycling has long been discussed. A work by Licata et al. was able to prove the formation of doxorubicinol or aglycones using human cytosolic fractions from myocardial samples.191 Structure-activity relations have suggested that aglycones and doxorubicinol may inflict cardiac damage causing oxidative stress or by perturbing iron homeostasis, respectively. The authors have proposed that the acute 29 ACS Paragon Plus Environment


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cardiac toxicity of doxorubicin should be attributed to the oxidant activity of aglycones, whereas the life-threatening chronic toxicity is related to alterations of iron and calcium homeostasis caused by doxorubicinol.191 In fact, that theory can help to explain the limited protective efficacy of antioxidants against chronic cardiomyopathy. Noteworthy, using murine models of acute chemotherapy-induced cardiac dysfunction, it was proven that antioxidants can be cardioprotective against the induced-oxidative stress of this regimen, 181, 185, 200 however they have failed to alleviate anthracycline cardiotoxicity in clinically relevant chronic animal models or clinical trials.196, 197 The initial actions of the semiquinones and aglycones cause a great burst of oxidative damage, but the alcohol metabolites formed are less redox active but largely accumulate in the heart and their actions cannot be attenuated by the antioxidants firstly administrated. The actions of the secondary alcohol metabolites go beyond the formation of reactive intermediaries, as they impair iron pathways and not just formation of ROS. The maintenance of a high and permanent antioxidant status by administration of conventional antioxidants is not possible at this point, due to their chronic toxicity, however the use of transgenic animals 178, 179, 201, 202 demonstrate that high and continuous cardiac antioxidant defenses would be important to alleviate anthracycline cardiotoxicity even chronic cardiomyopathy.201 In fact, in catalase overexpressing animals, the chronic heart damage caused by doxorubicin was significantly decreased .201 This suggests that low catalase activity in the heart may be an important factor responsible for the high sensitivity of the heart to doxorubicin-induced damage. Because catalase is a major enzyme that metabolizes H2O2 in the heart and this enzyme has no ability to react with doxorubicin and/or its metabolites, these results provide direct evidence to support the oxidative injury hypothesis for doxorubicin cardiotoxicity. Acknowledging that medications used to other forms of cardiomyopathy, particularly angiotensin-converting enzyme inhibitors and β-blockers, can be highly effective and may possess antioxidant properties, they can be an alternative to conventional antioxidant or genetic therapy.203 The use drugs with antioxidant proprieties with known pharmacokinetics, high cardiac distribution in the heart, and low toxicity, that impair other mechanisms involved in heart failure may be more efficient than purely antioxidant molecules. Carvediol has been proven valuable.204, 205 Pediatric patients with idiopathic dilated cardiomyopathy treated for 12 months with carvedilol were found to have significant improvements in LVEF and symptoms of heart failure. In fact, twelve months of carvedilol therapy was associated with antioxidant enzyme activities near those observed in healthy children. 205


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On the other hand, the protection offered by iron chelators may result mainly from counteracting doxorubicinol actions on iron homeostasis.191 In 2003, using a mice model of hemochromatosis, Miranda and colleagues demonstrated that the animals with hereditary hemochromatosis and treated with doxorubicin had a higher degree of cardiac mitochondrial damage and iron deposits than wild-type mice. Therefore, iron metabolism impairment largely contributes to doxorubicin cardiotoxicity.206 This theory is particularly popular because of the protective cardiac effects of dexrazoxane (an iron chelating agent), 207 however studies with chelators far more selective for iron than dexrazoxane, have also yielded negative or, at best, mixed outcomes,190, 207, 208 revealing that other mechanisms may be involved in dexrazoxane protective actions. The cardiotoxicity of doxorubicin, although related with oxidative stress, has probably many players and it is likely multifactorial. The oxidative stress hypothesis towards anthracyclines-induced cardiotoxicity has probably two stages both oxidative stressrelated: the first occurs mainly through the formation of semiquinones (one-electron reduction) or aglycones that promote an initial high oxidative stress burst; while the second stage refers to chronic cardiotoxicity, where the less redox active but still highly toxic secondary alcohols play a major role. The former can be interrupted by conventional acutely administered antioxidant therapy, while the later stage requires a more wide approach, with maintenance of high antioxidant status and iron chelation. Oxidative stress, ion deregulation, and concomitant alterations of mitochondria or of the cardiac-specific gene expression program eventually conspire to the cardiomyopathy caused by anthracyclines 169, and probably all the metabolites formed contribute at different stages to the cardiomyopathy-induced by anthracyclines.

2.4.2. Mitoxantrone

Mitoxantrone, an aminoanthraquinone synthesized in 1979 (Figure 4), belongs to a chemical class of agents known as anthracenediones whose clinical studies began in the United States in the 80’s.209 Mitoxantrone is used in prostatic and metastatic breast cancer, several types of lymphomas, and acute leukemia in therapeutic cycles (several administrations separated by drug-free weeks).210 Furthermore, mitoxantrone immunosuppressant characteristics enabled its approval in 2000 by the Food and Drug Administration for the treatment of worsening relapsing–remitting multiple sclerosis, secondary progressive multiple sclerosis, and progressive-relapsing multiple 31 ACS Paragon Plus Environment


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sclerosis.211 Mitoxantrone-induced cardiotoxicity is related to the total life-span cumulative dose and other factors similar to those referred in anthracyclines.209, 212 Mitoxantrone causes cardiotoxicity in a high number of patients, with decreased LVEF or development of congestive heart failure and dilated cardiomyopathy with similar clinical settings as anthracyclines.170, 210 The recommended maximum lifetime cumulative dose of mitoxantrone is 140 mg/m2, however several reports of cardiotoxicity occurred in multiple sclerosis treated patients, where the cumulative dose is usually lower. In fact, cardiac toxicity has been reported after therapy with mitoxantrone in cumulative doses as low as 100 mg/m2.170, 210, 211 As early as 1984, the cardiotoxicity of mitoxantrone was detected in 34 patients with advanced breast cancer that never received previous chemotherapy. They were treated with mitoxantrone (14 mg/m2 i.v. every 21 days).213 Before starting the mitoxantrone therapy, and every 3 months thereafter, radionuclide assessment of ventricular performance was obtained at rest and in response to stress. Ten patients showed a deterioration in ejection fraction, two of them developed congestive cardiac failure.213 Also in the 1980’s, 18 patients receiving mitoxantrone were evaluated with noninvasive tests for left ventricular function and by endomyocardial biopsy.214 The echocardiograms and systolic time intervals demonstrated a trend to cardiac deterioration that did not achieve statistical significance. However, the nuclear angiographic ejection fraction significantly decreased after 48 mg/m2 of mitoxantrone. The endomyocardial biopsies revealed tubular swelling, degeneration of mitochondria, minimal chromatin clumping, and myofibrillar lysis.214 The most recent published studies of mitoxantrone cardiotoxicity are related to multiple sclerosis treatments where lower mitoxantrone doses are recommended and no other concomitant cardiotoxic agents are usually administered. A retrospective study included all patients (163 patients) with multiple sclerosis treated with mitoxantrone at the University of British Columbia MS Clinic from 1999 to 2007.172 The standard treatment protocol consisted of mitoxantrone (12 mg/m2 i.v.) infused over 30 minutes, preceded by methylprednisolone (1 g i.v.) and dolasetron (100 mg i.v.).172 Mitoxantrone was administered monthly for 3 months, and then 3-monthly to a maximum cumulative dose of 120 mg/m2. By the end of the study, 14% developed de novo cardiotoxicity as measured by decreased LVEF.172 The underlying mechanisms of mitoxantrone cardiotoxicity are poorly understood. Since doxorubicin and mitoxantrone share clinical aspects of cardiac toxicity, their mechanisms were usually considered similar. However, the cardiotoxic mechanisms seem to differ in several aspects, namely mitoxantrone has a lower ability to enter in 32 ACS Paragon Plus Environment

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futile redox cycling. Mitochondria dysfunction can be a major source of oxidative stress in the heart 1 and mitoxantrone seems to greatly interfere with the bioenergetics of the heart 215 and then elicit mild oxidative stress. Oxygen uptake in rat heart slices was measured in a Warburg manometric apparatus over a period of 60 min.216 Data showed that the rate of oxygen uptake decreased with time at all concentrations (5-20 µM) except for the lowest concentration of mitoxantrone (5 µM). Mitoxantrone, as doxorubicin, inhibited cellular respiration and impaired cardiac respiratory control, but mitoxantrone was the most powerful in promoting ATP depletion (86% to control).216 Also, in neonatal rat heart myocytes, the levels of ATP were quickly disturbed by mitoxantrone.217 This energetic impairment can result of mitochondrial dysfunction and some degree of oxidative stress. In fact, in H2c9 cells incubated with mitoxantrone, an increase in the levels of ROS was only detected after ATP and ATP synthase compromise.215 In another study, using the same cellular model, mitoxantrone triggered the production of ROS, although the levels detected were substantially lower than those observed with doxorubicin.177 In primary cultures of cardiomyocytes from adult rats, mitoxantrone’s high lipophilic character was demonstrated to be an important factor in drug-induced acute cardiotoxicity. Mitoxantrone was found to reduce cell viability and number of rodshaped cells to the greatest extent, followed by carminomycin, idarubicin and epirubicin.218 This study clearly demonstrates that mitoxantrone lipophilicity warrants high concentration in the heart after to its administration 219, therefore being able to elicit high cardiac damage even if its ability to promote cardiac oxidative stress is low. Indeed, the role of lipid peroxidation in its cardiotoxic mechanisms seems to be of low relevance in mitoxantrone.220, 221 However, in HL-1 cells, we have showed that mitoxantrone (1 and 10 µM) was able to increase GSSG levels at 24 and 48h incubation, while total glutathione levels were significantly decreased at 48h with 10 µM.222 This in vitro study shows that mitoxantrone has the ability to impair antioxidant defenses. One aspect still poorly characterized in mitoxantrone is the involvement of iron on its cardiotoxicity. Mitoxantrone, at a low concentration of 2.5 µM increased the levels of ferritin, through a precondition state that requires previous formation of ROS.177 Studies using animal and isolated neonatal rat heart myocytes showed that the use of dexrazoxane in mitoxantrone-treated animals may be cardioprotective.223 In humans, an open-label study was performed to evaluate possible subclinical cardiotoxicity related to oxidative stress via Fenton reaction in multiple sclerosis patients treated quarterly with mitoxantrone (48mg/m2 cumulative dose) with and without concomitant dexrazoxane. No patient experienced symptoms of heart failure and patients receiving 33 ACS Paragon Plus Environment


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dexrazoxane, which is cardioprotective for anthracyclines, exhibited a significantly lower decline in LVEF.224 This protective role of dexrazoxane shows the pro-oxidant ability of mitoxantrone in the heart via iron pathways.224 Mitoxantrone is able to generate reactive species either through interactions with flavins or through the formation of a complex with Fe3+; however, such reactivity appears to be lower than that of doxorubicin.225 The apparent lower ability to produce reactive species referred earlier, may in fact be counterbalanced by its high entrance in healthy organs, 177 namely the heart 219 where mitoxantrone is highly accumulated. The pharmacokinetics and metabolism of mitoxantrone in man are still under debate, but they can have an important role in the drugs’ toxicity and its ability to disrupt the intracellular antioxidant and pro-oxidant equilibrium.215, 226-228 After a mitoxantrone administration of 14 mg/m2 as an i.v. infusion over 30 minutes, four metabolites were separated in human urine, the major metabolite being the dicarboxylic acid of mitoxantrone 219 (Figure 4). A few years later, the monocarboxylic acid of mitoxantrone was also identified as one major urinary mitoxantrone metabolite.229 Investigation on the biotransformation of mitoxantrone led to the discovery of another urinary metabolite in humans formed by the oxidation of the 1,4-dihydroxy-5,8-bis-alkylaminoanthraquinone moiety of mitoxantrone to an electrophilic intermediate followed by intramolecular nucleophilic attack of the secondary amino function in the side chain.226 This naphthoquinoxaline metabolite of mitoxantrone has been identified in animal and human models 226, 230 (Figure 4). In fact, we have recently demonstrated that this metabolite is present in the heart of rats exposed to mitoxantrone.230 This naphthoquinoxaline metabolite has been described to cause ATP decline in cardiac myocytes, being this effect reversed by dexrazoxane in higher extent than what was observed for mitoxantrone.217 In the presence of high concentration of H2O2 or by enzymatic activation through microsomal or peroxidase enzymes, the metabolism of mitoxantrone may involve oxidation, 226, 228 that can be followed by conjugation with GSH or glucuronic acid.230, 231 In fact, in the presence of GSH, the intermolecular nucleophile addition of the thiol group results in the formation of the thioether conjugates of mitoxantrone.227 Whereas the intramolecular attack of the nucleophile side chain N-atom on the oxidized mitoxantrone takes place at position C-6 leading to a naphthoquinoxaline metabolite of mitoxantrone, the corresponding reaction with cellular nucleophiles, namely GSH, seems to occur at the dihydroxy-substituted six-membered ring.227, 230 This conjugation can lead to a disequilibrium towards a decrease in cardiac antioxidant defenses.227 Although the heart has poor overall peroxidases activity, the fact remains that the heart


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has a high basal formation of H2O2 even in non-stressful situations, thus the reaction of activated mitoxantrone with GSH can be possible in vivo. In conclusion, mitoxantrone can be less prone to create a pro-oxidative state when compared to doxorubicin, and several contradictory studies relating mitoxantrone and oxidative stress are found in the literature. Concentrations, models and mainly time points seem to be the major handicaps in the evaluation of mitoxantrone oxidative stress potential. In the authors’ point of view, several conditions are of outmost relevance to mitoxantrone-induced oxidative stress in the heart. Although mitoxantrone is more resistant to reductive enzyme activation, it is a good substrate to enzymatic oxidation, being the redox active metabolites able to form low amounts of ROS but are capable of binding to intracellular nucleophiles, like GSH. Mitoxantrone and specially its metabolites are able to cause slight impairments of GSH/GSSG ratio, 230 probably causing initial insidious alterations in the antioxidant balance. However the energetic impairment caused by mitoxantrone and metabolites is, in the long run, the major contributor to oxidative stress and iron seems to be involved.177, 217 The work by Shipp et al. demonstrates that the metabolite naphthoquinoxaline has a higher potential on ATP depletion than mitoxantrone and importantly that the bioenergetics impairment hugely depends on iron availability. Furthermore, the catechol ring of mitoxantrone and of the metabolite naphthoquinoxaline seems to be relevant to the observed ATP decrease, since ametantrone (that does not have that ring) has no significant effect on ATP levels.215 One can speculate by putting together all the scarce data available that mitoxantrone is not able in vivo to cause an initial redox cycle that is sufficient to cause an early and abrupt oxidative stress (as do the aglycones and semiquinones of doxorubicin), however it (and its naphthoquinoxaline metabolite) rapidly accumulates in the heart 219, 230 easily impairing ATP homeostasis in the heart, factors that seem to depend on iron.217 In fact, mitoxantrone probably shares more characteristics of doxorubicinol cardiotoxicity than of doxorubicin semiquinone, as mitoxantrone and doxorubicinol share the high cardiac accumulation and interference with iron homeostasis.215, 217, 230

2.4.3. Cyclophosphamide

Cyclophosphamide, an alkylating agent, can produce heart failure or myocarditis. These symptoms usually present themselves in the first weeks of therapy. 232 Cyclophosphamide is rarely cardiotoxic at low/standard doses but it can cause severe 35 ACS Paragon Plus Environment


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cardiotoxicity when administered at high doses.233, 234 High-dose cyclophosphamide is commonly integrated in the mobilization regimen of patients undergoing autologous hematopoietic stem cell transplantation.232, 235 High therapeutic doses of cyclophosphamide may cause a lethal cardiotoxicity. A combination of symptoms and signs of myopericarditis can result in congestive heart failure, arrhythmias, cardiac tamponade, and myocardial depression. 236 The incidence of fatal cardiomyopathy varies from 2.0% to 17.0%, depending on the different regimens and populations. In contrast to the anthracycline-induced cardiomyopathy that occurs months to years after a high cumulative dose, cyclophosphamide-induced cardiomyopathy occurs within the initial 2 or 3 weeks after treatment.236 In the work of Goldberg and co-workers with patients that had not received any other cardiotoxic therapy namely anthracycline or radiation 234, an incidence of congestive heart failure was reported to be 0/32 in patients receiving doses lower than 1.55 g/m2 and 6/52 in patients receiving higher than 1.55 g/m2 cyclophosphamide.234 Reversible decreases in electrocardiogram voltage and increases in left ventricular mass, possibly reflecting myocardial edema or hemorrhage were observed in the twice-daily, higher-dose (mean total 174 +/- 34 mg/kg) of cyclophosphamide regimen. 234 In a work by Zver et al. highdose cyclophosphamide (4 g/m2) was used in 23 consecutive multiple myeloma patients.237 Significant neurohumoral activation was seen through an increase in plasma brain natriuretic peptide (BNP) and endothelin-1 levels. Left ventricular diastolic dysfunction occurred early after the cyclophosphamide regimen and mild functional mitral regurgitation was observed in patients given high-dose cyclophosphamide therapy.237 In vivo studies conducted in laboratory animals showed that cyclophosphamide cardiotoxicity is oxidative stress related. A single dose of cyclophosphamide (200 mg/kg, i.p.) administrated to adult male Wistar albino rats caused down regulation of antioxidant genes (GPX, catalase, and SOD), increased lipid peroxidation, and decreased the ATP (40%) and ATP/adenosine diphosphate (ADP) ratio (44%) in cardiac tissues.238 Corroborating these data, adult male Wistar rats given the same dose had a significant decrease in the levels of enzymes (SOD, catalase, GPX, GR and GST) and non-enzymatic antioxidants (GSH, vitamin C, and vitamin E) along with high malondialdehyde levels.239 These findings highlight the pro-oxidant aspects of cyclophosphamide cardiotoxicity thus explaining its synergistic effect on the cardiotoxicity of anthracyclines or mitoxantrone. It is nowadays accepted that several therapeutic and toxic effects of cyclophosphamide seem to require its metabolic activation by hepatic microsomal cytochrome P450 mixed function oxidase system 240 36 ACS Paragon Plus Environment

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and those metabolites in vitro and in vivo were shown to cause cardiotoxicity through oxidative stress mechanisms.241-245 Cyclophosphamide, acrolein (a metabolite of cyclophosphamide), and doxorubicin were tested in the H9c2 cell line.245 Cyclophosphamide by itself had no toxic effect, whereas the toxicity of acrolein was 1000 times higher than that of doxorubicin in the H9c2 cell line. Also, in isolated Langendorff-perfused rat hearts, acrolein, but not cyclophosphamide, reduced the left ventricular developed pressure and heart rate, and increased the left ventricular end diastolic pressure.245 Neonatal rat heart myocytes were used to evaluate the acute cardiotoxic effects of cyclophosphamide metabolites.241 The myocytes had similar results on ATP levels depletion with 4-hydroperoxycyclophosphamide and acrolein with 50% inhibitory concentrations (IC50) of 123 µM and 152 µM, respectevely.241 The parent compound, cyclophosphamide had no significant action on ATP levels. GSH levels were initially reduced and then transiently elevated following exposure to hydroperoxycyclophosphamide.241 With acrolein, GSH levels were reduced to nonmeasurable levels at all-time points evaluated (up to 72h). Finally, two exogenous sulfhydryl-containing compounds (mesna and N-acetylcysteine) were shown to block the toxic effects of both 4-hydroperoxycyclophosphamide and acrolein. These results suggest that acrolein and hydroperoxycyclophosphamide are equipotent cytotoxins and that a transient depletion in GSH accompanies their toxic effects in cardiac myocytes. 241

Adult mouse cardiomyocytes exposed to 1 µM of acrolein showed a marked

increase in the intracellular ROS and calcium concentration, by 12- and 2-fold, respectively, compared to control values.244 Furthermore, exposure to acrolein caused inflammation and cardiomyopathy in C57BL/6 mice.243 Histological and biochemical evaluation revealed myocardial oxidative stress (membrane-localized protein-4hydroxy-trans-2-nonenal adducts) and nitrosative stress (increased proteinnitrotyrosine) in acrolein-treated mice. Acrolein also led to cardiomyocyte hypertrophy, increased apoptosis, and disrupted endothelial nitric oxide synthase in the heart 243, which can favor the formation of ONOO

. This study also demonstrated that

translocation to the heart of acrolein can occur, which is crucial step since cyclophosphamide metabolization occurs mostly in the liver.240 Cyclophosphamide effects on the heart have a rapid onset and occur after high dose regimen. The oxidative stress observed is related to the metabolites. Although the heart has a low ability to metabolize the parental molecule, the hepatic metabolization followed by metabolite distribution, warrants sufficient concentration of the toxic metabolites in a short period of time. At this point, hydroperoxycyclophosphamide and 37 ACS Paragon Plus Environment


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acrolein have proven actions on cardiac cellular defenses, namely glutathione, that seem to make them the main culprits of cyclophosphamide oxidative stress related cardiotoxic actions. Other pro-oxidative mechanisms, like inflammation, can also promote cardiac oxidative stress, although at this point scarce studies have been made.

2.4.4. Multiple drugs in anticancer therapy

Anthracyclines and other high-dose chemotherapeutic agents represent the greatest risk for cardiotoxicity, but the use of multiple cardiotoxic agents results in relevant synergistic effects, even when, the cumulative total dose of each agent is considered to be safe (Table 1). Nineteen adult patients were pretreated with conventional chemotherapy preparative regimen and hematopoietic cell transplantation in acute leukemia containing idarubicine, daunorubicine, and mitoxantrone. The standard doses for a cycle of chemotherapy were: idarubicine 3 x 12 mg/m2, daunorubicine 3 x 50 mg/m2, mitoxantrone 3 x 10 mg/m2.246 In all patients, the myeloablative preparative regimen consisted of i.v. cyclophosphamide in a total dose of 120 mg/kg; in 13 of the patients a combination with per os busulphan 16 mg/kg was used, and in 6 patients fractionated total body irradiation was done. After the cytotoxic treatments, hematopoietic cell transplantation followed. Plasma N-terminal prohormone of brain natriuretic peptide (NT-proBNP), cardiac troponin T (cTnT) and creatine kinase-MB (CK-MB) mass concentrations were measured on the day before the myeloablative preparative regimen, the day after myeloablative preparative regimen, the day after hematopoietic cell transplantation and, also, 14 days after hematopoietic cell transplantation. Of note, it has been reported that normal plasma BNP/NT-proBNP concentrations practically exclude heart failure due to high negative predictive value of the test,247 while cardiac plasma troponins — cardiac troponin T (cTnT), cardiac troponin I (cTnI) — and myocardial isoenzyme of creatine kinase (namely CK-MB) are cardiospecific markers that show structural damage of cardiomyocytes.248 Before the preparative regimen, mean plasma NT-proBNP values were 106.3 ± 55.7 ng/l, but after, they increased to 426.1 ± 391.5 ng/l. After hematopoietic cell transplantation, they further increased to 847.6 ± 780.6 ng/l, while 14 days after transplantation, the mean NT-proBNP values were 330.8±236.8 ng/l. The values were statistically significant in comparison with the baseline values. In all patients, plasma cTnT and CK-MB mass concentrations 38 ACS Paragon Plus Environment

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remained unchanged during the time period of the evaluations. NT-proBNP elevations in 12 (63.2 %) patients were persistent, indicating subclinical cardiotoxicity (risk for the development of heart failure). Higher NT-proBNP elevations in patients with higher cumulative doses of anthracyclines (>450 mg/m2) and in patients with preparative regimen containing a combination of high-dose cyclophosphamide and body irradiation confirm that these therapeutic procedures are more cardiotoxic.246 Also, early clinical and subclinical cardiotoxicity is also frequent in Non-Hodgkin's lymphoma patients receiving the usually designated CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisolone). In these cases, the threshold cumulative dose for cardiotoxicity of doxorubicin appears to be low: at doses >200 mg/m2, 27% of patients showed cardiac dysfunction.249 These studies and also the reports summarized in Table 1 show that the combination of several compounds trigger synergist cardiotoxic effects. When referring to the combination of mitoxantrone, anthracyclines and/or high doses of cyclophosphamide, the formation of highly reactive metabolites with prooxidant effects and the impairment of antioxidant defenses favor cardiotoxicity and demonstrate the heart’s susceptibility towards oxidative stress. When referring to inhibition of HER2 /tyrosine kinases or in the case of paclitaxel, the use in combination also favors cardiotoxicity while interfering with antioxidant defense adaptation mechanisms (i.e. trastuzumab) or by favoring accumulation of highly reactive molecules (i.e. taxanes). The inhibition of tyrosine kinase activity has markedly improved the management of cancers including chronic myeloid leukemia, breast cancer, gastrointestinal stromal tumor, renal cell carcinoma, and colon carcinoma. Inhibitors of tyrosine kinases are subdivided in 2 classes: humanized monoclonal antibodies, typically targeting growth factor receptor tyrosine kinases, and small molecules, referred to as tyrosine kinase inhibitors (TKIs), targeting both receptor and nonreceptor tyrosine kinases.250 HER2 is a proto-oncogene and member of the ErbB (epidermal growth factor receptor) family of transmembrane tyrosine kinases 251 (Figure 5). Approximately 15%-25% of patients with breast cancer have tumors that overexpress the HER2 protein or amplify the gene. The HER-2 amplification has a significant negative predictor of both overall survival and time to relapse in patients with breast cancer.174 Current adjuvant treatment for HER2-positive tumors often includes trastuzumab, a recombinant monoclonal antibody against HER2, either concurrent with or sequential to systemic chemotherapy. The addition of trastuzumab to standard adjuvant chemotherapy yielded a significant improvement in both disease-free survival and overall survival compared with chemotherapy alone.252, 253 However, the incidence of trastuzumab-induced 39 ACS Paragon Plus Environment


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cardiotoxicity hugely increases in combination therapy, being usually overshadowed by the substantial improvements in disease-free and overall survival. Salmon et al. described a phase 3 clinical trial with women whose cancer overexpressed HER2 where several groups were formed.254 Two hundred and thirty four patients received standard chemotherapy alone and 235 patients received standard chemotherapy plus trastuzumab. Patients that had not previously received adjuvant (postoperative) therapy with an anthracycline were treated with doxorubicin (or epirubicin in the case of 36 women) and cyclophosphamide with (143 women) or without trastuzumab (138 women). Patients who had previously received adjuvant anthracycline were treated with paclitaxel alone (96 women) or paclitaxel with trastuzumab (92 women). Trastuzumab increased clinical benefit of first-line chemotherapy in metastatic breast cancer that overexpresses HER2, but also increased the amount of cardiac dysfunction detected. Cardiac dysfunction, symptomatic or asymptomatic, occurred in 27% of women given an anthracycline, cyclophosphamide, and trastuzumab in combination; 8% in the group given an anthracycline with cyclophosphamide; 13% in the group given paclitaxel and trastuzumab; and 1% in the group given paclitaxel alone.254 This pivotal trial by Slamon and colleagues administrating trastuzumab combined with chemotherapy in metastatic breast cancer showed an enhancement in the incidence of congestive heart failure among patients who simultaneously received trastuzumab and anthracycline-based therapy.254 Several other randomized clinical studies were done with trastuzumab, some of them done in real world scenarios, showing higher prevalence of trastuzumab cardiotoxicity, but these reports had more variables involved.253, 255 In addition to their expression in certain tumors, HER receptors are expressed in the myocardium where they serve as receptors for neuregulins.256 HER2 signaling is essential for cardiomyocyte survival since binding of neuregulin-1 initiates cell survival pathways, inhibits apoptosis and maintains cardiac function, namely by the up regulation of MAPK/extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2), phosphoinositide 3 kinase/AKT and by the activation of focal adhesion kinases. These two latter mechanisms stabilize myofibril structure and contribute for cardiomyocytes’ survival.257 Neuregulin-1 signaling activates the phosphoinositide 3 kinase/AKT signal transduction cascade that is able to initiate a change in mitochondrial respiration and thereby decrease the production of ROS.257 Moreover, in freshly isolated adult primary rat cardiomyocytes treated for 24 h with recombinant human neuregulin-1beta, the upregulation of redox factor proteins thioredoxin and the thioredoxin reductase 1, and GSTP1 was found.256 Trastuzumab has documented cardiotoxic effects, because in the myocardium it binds to HER2 with high affinity, thereby eliminating its ability to dimerize with other HER receptors.253, 257 By inhibiting HER2’s ability to dimerize and activate its’ 40 ACS Paragon Plus Environment

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signaling pathways, cardiomyocytes are unable to cope with additional stress. Trastuzumab blocks this activation and, via multiple mechanisms including alterations in levels of Bcl-X family members, leads to mitochondrial dysfunction, energy compromise, and cytochrome c release.250 In primary cultures of neonatal rat ventricular myocytes, HER2 inhibition by trastuzumab is associated with a dramatic increase in expression of the pro-apoptotic Bcl-2 family protein Bcl-xS (which is generally not expressed in a normal cardiomyocyte) and a decreased in the levels of anti-apoptotic Bcl-xL (which is normally over-expressed). These alterations cause significant mitochondrial dysfunction and are associated with a 35% decline in ATP levels (i.e. disruption of cardiomyocyte cellular energetics).258 In fact, antibody blockade of the HER2 receptor caused cardiomyocyte death through mitochondrial and ROSdependent pathways, which were reversed by the antioxidant, N-acetylcysteine. The effects of HER2 antibody on both cell death and ROS production were also reversed by cyclosporine A and diazoxide, chemicals that regulate the pro- and anti-apoptotic channels in the mitochondria, respectively.259 These in vitro experimental models demonstrate the correlation between mitochondrial dysfunction and oxidative stress in cardiomyocytes blocked by HER2 antibody. The high number of dysfunctional cardiac mitochondria results in high production of O2●− that gathered with the low antioxidant levels of the heart, results in oxidative stress. Anthracycline-trastuzumab-containing regimens demonstrate significant clinical efficacy in HER2-positive breast cancer; however, this strategy is limited when unacceptable cardiotoxicity occurs, particularly with their concurrent administration. The toxicity observed is mainly related to the ability of both compounds to interfere with different oxidative stress mechanisms. Neuregulin and the antioxidant N-acetylcysteine, protected against doxorubicin oxidative-induced damage in adult rat ventricular cardiomyocytes.179 Cardiomyocytes isolated from mice with a ventricular-restricted deletion of HER2 are more susceptible to pro-oxidant anthracycline toxicity 260 as more ROS will accumulate due to the inhibition of the essential MAPK family, namely the extracellular signal-regulated protein kinase 1/2 cell survival pathway (Figure 5A).

257

Moreover, neuregulin and doxorubicin showed an opposite modulation of glutathione reductase gene expression.179 The inhibition of HER2 resulted in a minor increase in oxidative stress in adult ventricular cardiomyocytes, 261 not being a critical aspect towards heart toxicity; however trastuzumab partly blocks the protective effect of neuregulin activation upon the antioxidant defenses of the myocardium (Figure 5B). On the other hand, anthracyclines largely increase oxidative stress in the heart and they also interfere with HER2 pathways. In fact, shortly after completion of anthracycline 41 ACS Paragon Plus Environment


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treatment, myocardial HER2 over-expression was detectable in 50% of the patients and those values were gradually reduced over a variable period after cessation of anthracycline treatment.262 That apparent attempt to activate survival pathways in the heart is blunted if trastuzumab is given immediately after anthracycline cycle, increasing cardiac dysfunction. Thus nowadays to decrease the incidence of cardiac adverse events in combination therapy, anthracyclines and trastuzumab should be administered in a sequential manner. Other studies have linked the renin-angiotensin system, including angiotensin II signaling, to doxorubicin and trastuzumab-induced cardiac dysfunction through the alteration of NADPH oxidase and MAPK signaling pathways.263 These alterations of HER2 signaling through NADPH oxidase and MAPK are connected to increase in oxidative stress.36 To circumvent the problems found in antibodies and for a more specific tyrosine kinase inhibition (and therefore lower cardiotoxicity), new molecules that inhibit tyrosine kinase signaling were synthesized since tyrosine kinase signaling is essential to both malignant transformation and tumor angiogenesis (for a broad revision on tyrosine kinase inhibitors cancer therapeutics see Chen et al. 2008). 250 The use of small molecules that both inhibit the receptors involved in neuregulin pathways and/or the several intracellular mechanisms activated, brought a new set of problems regarding cardiotoxicity. Depending on their place of action and on the studies available until this point, tyrosine kinase inhibitors have unknown cardiovascular toxicity or moderate cardiovascular toxicity (e.g. sunitinib or dasatinib).250 The mechanisms are variable among tyrosine kinase inhibitors, but to the best of the authors’ knowledge very few studies regarding these molecules and oxidative stress in cardiac models have been made so far. Sunitinib treatment of neonatal rat myocytes caused a dose-dependent damage at clinically relevant concentrations.264 Dexrazoxane, which is a clinically approved doxorubicin cardioprotective agent, did not protect myocytes from damage, which suggests that sunitinib does not induce oxidative damage via modifying iron pathways.264 The lack of data at this point does not allow any assumption regarding the tyrosine kinases inhibitors and oxidative stress-related cardiotoxicity. Taxanes (e.g. paclitaxel, docetaxel) are mostly used in combination with other chemotherapeutics to improve survival rate on cancer patients.254, 265, 266 The incidence of cardiac toxicity with taxanes is low when used as a single agent. However the reported cardiotoxicity is synergistically increased when taxanes are used in combination with other agents that have known cardiotoxicity (Table 1). Paclitaxel 42 ACS Paragon Plus Environment

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causes arrhythmias or blood pressure disorders, being these manageable effects attributed to the vehicle cremophor EL. In a phase 3 randomized trial, docetaxel, doxorubicin, and cyclophosphamide were given every 3 weeks for six cycles.267 Heart failure occurred in 3% of those patients and a substantial decrease in left ventricular ejection fraction (defined as a relative decrease from baseline of 20% or more) was seen in 58 (17%) patients treated with that regimen.267 In a smaller study in 1996 conducted in women with advanced breast cancer, the use of doxorubicin and paclitaxel soon after, caused a significant decrease in left ventricular ejection fraction to below normal levels in 50% of patients while 20% of them developed congestive heart failure. 268 Paclitaxel has a low ability to promote oxidative stress in isolated heart cells 261, thus it is highly unlikely that the synergistic cardiotoxic effects of the combination with anthracyclines is related to its ability to generate oxidative stress. The pharmacokinetic interaction between those molecules (taxanes and anthracyclines) is the most plausible reason to explain the increased toxicity of the mixture. A pharmacokinetic study showed that paclitaxel enhances the nonlinearity of doxorubicin pharmacokinetics and significantly decreases the systemic clearance of both doxorubicin and doxorubicinol. 269

The paclitaxel/doxorubicin interaction was found to be paclitaxel-dose dependent,

doxorubicin concentration-dependent, and may be the result of competition for elimination mechanisms, possibly competition for hepatic and biliary transporter proteins such as p-glycoprotein. In fact, the formulation vehicle of paclitaxel, polyethoxylated castor oil (cremophor EL), has been associated to the inhibition of those transporters.269 Therefore, the heart is exposed to higher concentrations of the parent anthracycline and its metabolite. Furthermore, paclitaxel (and not the vehicle) and docetaxel are implicated in allosteric interactions with cytoplasmic aldehyde reductases, increasing their activity. Cytoplasmic aldehyde reductases are implicated in doxorubicinol formation in the human heart as previously discussed.270 In isolated human heart cytosol or in myocardial strips, paclitaxel and docetaxel caused allosteric effects upon reductases, thus stimulating the formation of doxorubicinol, but not of epirubicinol, when exposed to the same concentrations of the parent anthracyclines and taxane-cosolvent formulations.271 These data showed dissimilar aspects between doxorubicin/epirubicin when regarding taxane interactions. However, in a work by Esposito and collaborators, the plasma metabolites of epirubicin, epirubicinol and its aglycone, increased significantly with concomitant administration of paclitaxel in humans.272 This study conducted in humans showed the overall contribution of two important metabolic pathways of anthracyclines, reductive deglycosidation and carbonyl reduction, demonstrating that taxanes are able to increase the secondary 43 ACS Paragon Plus Environment


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alcohol metabolite of epirubicin and its aglycone,272 while the in vitro study only took into account the contribution of the cardiac metabolization.271 Furthermore, in the human cardiac fractions, equimolar concentrations of anthracyclines showed that epirubicin was able to produce no more than one-third of the amount of the corresponding secondary alcohol metabolite when compared to the amount of doxorubicinol produced by doxorubicin. 271 Different amounts of taxanes/epirubicin may be required to obtain observable effects on epirubicinol’s formation, perhaps due to the low ability formation of epirubicinol in vitro. Altogether, these results uncover the fact that anthracycline cardiotoxicity is aggravated by any concomitant drug that stimulated the formation of secondary alcohol metabolites or even aglycones that, as stated before, are highly toxic to the heart. However, this theory does not fully explain the high cardiotoxicity of taxanes with trastuzumab. As seen by Slamon et al. in 2001, heart problems hugely increased just by adding trastuzumab to paclitaxel.254 In primates and in humans a 1.5-fold increase in mean serum concentrations of trastuzumab occurs when paclitaxel is given. This decrease in trastuzumab clearance can explain the synergistic effects on cardiotoxicity of this drug combination.273 The synergistic cardiotoxic effects of combination anticancer therapy are evident in the clinical setting, and oxidative stress is a main culprit in several cases. The mixture of chemotherapeutics can promote pharmacokinetic interactions with higher accumulation of pro-oxidant parenteral molecules or intermediates in the heart (anthracyclines and taxanes) or by lowering cardiac antioxidant defenses (trastuzumab, mitoxantrone or cyclophosphamide), thus impairing cardiac function.

3. Conclusions

The compounds herein presented share at least one of the two following characteristics: 1) interfere with cellular redox homeostasis, because they (or their metabolites) are chemically reactive compounds that undergo redox cycling with formation of ROS or bind to cellular antioxidant nucleophiles; or 2) interfere with mitochondria homeostasis making the electron chain transporter ‘leaky’, thus promoting oxidative stress. Most of the molecules referred in this review promote those two mechanisms in different proportions, but the two are equally lethal to the heart.


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Several of the molecules addressed herein as cardiotoxicants are prone to promote oxidative stress, or to disrupt cardiac organelles or enzymes through the formation of electrophilic metabolites which are highly reactive species. In fact, when observing the molecular structures of these drugs, most of them are able to enter a futile and cellular damaging redox cycle. The formation of quinones, namely in doxorubicin and catecholamines, the involvement of iron, referred for mitoxantrone and anthracyclines, the formation of highly reactive metabolites in ethanol and cyclophosphamide (and perhaps mitoxantrone), the onset of a catecholaminergic surge in the drug abuse scenario (although several studies advocate the direct reactivity of those drugs of abuse or their metabolites towards the heart), all result in cardiac oxidative damage. Furthermore, xenobiotics that increase the activity of the electron transport chain or make it more ROS-‘leaky’ can be equally major contributors to oxidative stress in the myocardium, as seen in mitoxantrone. The rational therapy for these oxidative stress related complications should therefore be antioxidant supplementation. The antioxidant ‘therapy’ used to decrease the injury caused by oxidative stress related cardiotoxicants revealed controversial results since exogenous administered antioxidants have failed to show therapeutic benefits in several classes while others advocate for protective actions. Actually, the exogenous antioxidants are short lived and sometimes do not reach reasonable amounts in the heart.274 On the other hand, intrinsically, some chronic antioxidant supplementation has also pro-oxidant properties that can be damaging to the heart.275 In the case of exogenous administration of antioxidant enzymes, these large proteins cannot efficiently reach the intracellular medium, while smaller antioxidant molecules can easily reach the organ but transiently, since their elimination is also fast. In general, the studies performed to date in mice with genetically altered levels of antioxidant enzymes yield more consistent results that support the involvement of ROS in the mechanism of several cardiotoxicants 156, 178, 276-282 than studies that involve exogenous administration of antioxidants.283-286 This difference demonstrates a need for a substantial elevation of intracellular antioxidant levels for cardioprotection and especially for their continuous availability within the cells instead of the transient rise seen in the exogenous administered antioxidants. Moreover, one should keep in mind that the heart is a particularly redox sensitive organ that easily and quickly adapts in response to the body’s needs. The redox status of several molecules is the starting point for several pathways that lead to cardiac adaptation. Thus, lower levels of ROS activate particular signal transduction pathways, many involved in protector preconditioning signals.78, 287-290 In fact, the heart is able to 45 ACS Paragon Plus Environment


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activate several survival mechanisms when facing low levels of ROS, and those pathways are crucial for cardiac fate.78 The heart inability to achieve that initial adaptation can make it more vulnerable in a continuous injury. When administrating antioxidants, it is often forgotten that antioxidants fail to distinguish between the ROS and RNS that play a physiologic role and those that cause damage.274 Damage to the heart leads to compensatory mechanisms that include activation of the neurohumoral pathways (such as the renin-angiotensin and adrenergic systems), myocardial hypertrophy, and possibly survival pathways such as the neuregulin/epidermal growth factor receptor system.174 Therefore, the inhibition of pathways that are activated in heart injury may be a better protective strategy, especially when the therapeutic intervention also contains antioxidant proprieties. Most definitely, oxidative or nitrosative insults or energetic metabolism disruptions are the most common onsets to cardiac damage that lead to life threatening situations or the loss of cardiac work quality. Oxidative stress promoted by drugs can be the starting point of multifactorial cardiac diseases and definitively deserves further attention.

Funding sources: This work received financial support from ‘Fundação para a Ciência e Tecnologia (FCT)’, Portugal (EXPL/DTP-FTO/0290/2012) and by ‘Fundo Comunitário Europeu’ (FEDER) under the frame of ‘Eixo I do Programa Operacional Fatores de Competitividade (POFC) do QREN’ (COMPETE: FCOMP-01-0124-FEDER-027749). VMC thanks FCT for her Post-doc grant (SFRH/BPD/63746/2009).


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Legends of figures and tables

Figure 1 – Structural image of catecholamines and of drugs of abuse are known to contribute to cardiac oxidative stress.

Figure 2 – The mechanisms of cocaine induced toxicity via oxidative stress. Cocaine increases sympathetic output, leading to a huge release of noradrenaline (NA) and adrenaline (ADR) from the adrenal medulla. Cocaine is also a powerful central nervous system stimulant, and it also inhibits noradrenaline transporter (NET). These higher levels of catecholamines result in higher blood pressure, heart rate, and contractibility. Furthermore, cocaine has a potent action on sodium influx, it increases L-type calcium channel current, and blocks potassium channels, factors that favor arrhythmias.

Figure 3 – Doxorubicin easily enters cells through passive diffusion and accumulates in the cardiomyocytes. Doxorubicin accumulates in the mitochondria due to doxorubicin high affinity towards cardiolipin. This anthracycline may become cardiotoxic after oneor two-electron reductive activation. One electron reduction of the quinone moiety of doxorubicin results in the formation of a semiquinone free radical, which regenerates back to the parent quinone by reducing molecular oxygen to O2●_. The radical is converted to H2O2 spontaneously or through the action of SOD. H2O2 leads to hydroxyl radical (HO•) through Fenton reactions. The highly toxic HO• causes directly oxidative stress and contributes to mitochondrial damage. The NADPH-oxidoreductases [cytochrome P450 or -b5 reductases, mitochondrial NADH dehydrogenase, xanthine dehydrogenase, endothelial nitric oxide synthase (reductase domain)] can be responsible for that reduction in the heart. Two electron reduction of the carbonyl in the side chain results in the conversion of doxorubicin to the secondary alcohol doxorubicinol. Doxorubicinol is able to alter calcium homeostasis and promote iron release from its’ pools, thus contributing to oxidative stress. Doxorubicinol is more hydrophilic and accumulates within the cardiomyocyte. Doxorubicin can also suffer deglycosidation on the sugar moiety, forming aglycones (for simplicity only the main skeletal of aglycones is depicted). Aglycones are powerful mitochondrial toxins. Mitochondrial dysfunction further amplifies oxidative stress.

Figure 4 – Mitoxantrone’s most common metabolites. The major urinary metabolites of mitoxantrone are the mono- and the dicarboxylic acids of mitoxantrone. Another urinary metabolite (naphthoquinoxaline metabolite) is formed by the oxidation of the 1,4dihydroxy-5,8-bis-alkylamino-anthraquinone moiety of mitoxantrone to an electrophilic 62 ACS Paragon Plus Environment

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intermediate, which suffers intramolecular nucleophilic attack of the secondary amino function located in the side chain. In the presence of high concentration of H2O2 or after enzymatic activation through microsomal or peroxidase enzymes, the metabolism of mitoxantrone may involve oxidation, then followed by conjugation with GSH or glucuronic acid.

Figure 5 – (A) Neuregulin-1 binds to and activates Human epidermal growth factor receptor (HER) 4, which is then ready for dimerization with HER2. HER2 signaling is essential to cardiomyocytes’ survival because the binding of neuregulin-1 initiates cell survival pathways. Those pathways are: inhibition of apoptosis and maintenance of cardiac function, namely by up regulation of MAPK/ERK 1/2, the phosphoinositide 3 kinase/AKT signal transduction cascade, and activation of focal adhesion kinases. AKT is able to change mitochondrial respiration, by decreasing the production of ROS. AKT can also alter the expression of BCL-2 family proteins, initiate glucose uptake and trigger activation of endothelial nitric oxide (•NO) synthase. These changes have also been related to an increase in cell’s survival. (B) Trastuzumab binds to HER2 with high affinity, thereby eliminating its ability to dimerize with other HER receptors. By inhibiting HER2’s ability to dimerize, trastuzumab impairs the cardiomyocytes’ ability to activate any of the cell signaling survival pathways, namely MAPK/ERK 1/2, phosphoinositide 3 kinase/AKT, and focal adhesion kinases-dependent pathways. Cardiomyocytes are rich in mitochondria, thus after HER2 inhibition, cardiomyocytes produce more ROS and simultaneously are unable to cope with the added stress, being more susceptible to oxidative stress insults. Combination therapy with anthracyclines, cyclophosphamide or mitoxantrone clearly increases the oxidative stress-related heart damage seen with trastuzumab.

Table 1 – Reports of cardiotoxic regimens of multiple cytostatic drugs.



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Table 1 Cytostatic drug and

Potential cardiotoxic regimens

Bibliography

Cardiac problem

disease setting METASTATIC

BREAST

CANCER

TREATED

MOSTLY

WITH

Trastuzumab treatment was given to all patients. Median cumulative doses of doxorubicin or 2

2

patients

Some patients received also high dose therapy with mitoxantrone (16 patients > 60 mg/m-2),

patients

developed

291

presented

cardiac dysfunction; and 3.8%

2

PACLITAXEL-CONTAINING

of

epirubicin were 266 mg/m (range 60–500) and 395 mg/m (range 120–660), respectively.

thiotepa or/and melphalan. Cyclophosphamide (>4 g/m ) was administrated in 37 patients.

ANTHRACYCLINE/

17.9%

congestive

heart failure.

Radiation therapy on cardiac area was given some patients (16 patients) and in others previous prior anthracycline/paclitaxel (44 patients) was also given.

REGIMENS (N= 53). HER2-POSITIVE

BREAST

CANCER WOMEN (N=499)

Trastuzumab was administered in all centers at a loading dose of 8 mg/kg body weight i.v. once,

Cardiotoxicity was observed in

followed by maintenance doses of 6 mg/kg every 3 weeks for 1 year (18 total doses). Adjuvant

133 patients (27%): 102 (20%)

anthracycline-based chemotherapy, with or without taxane, had been administered in 87% of

showed asymptomatic reduction

patients and anthracycline- and taxane-based therapy in 49%. Of the 133 patients that exhibited

in LVEF of >10% but < or =20%;

cardiotoxicity, 90 % were subjected to therapy with anthracyclines. 69% of the patients with

15

registered cardiotoxicity were subjected to radiotherapy: 16% of these patients had doxorubicin in

asymptomatic decline of LVEF of

2

a median dose of 231 ± 46 mg/m . 75% of the anthracycline treated patients had epirubicin in 2

mean dose of 339 ± 156 mg/m . 61% of patients had been treated with radiotherapy after

patients

(3%)

253

had

>20% or

The Heart As a Target for Xenobiotic Toxicity: The Cardiac

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