**5. Cardiospecific toxicity of doxorubicin**

The clinical utilization of DOX is limited because of its toxic impact, especially on heart tissues, e.g., heart failure, cardiomyopathy [20]. The mortality rate of congestive heart failure induced by DOX is estimated at around 20%. There is no explanation for how DOX causes its toxicity on noncancerous tissue. However, it is thought to be multiple and complex mechanisms, nitrosative and nitrative stress, DNA damage, dysregulation of metabolites, and inflammation [16] involving DOX's toxicity, eventually triggering apoptotic cell loss [43]. The dysfunction of energy production has played a critical role in the development of both acute and chronic DOX toxicity and is related to time-dependent mitochondrial dysfunction [43]. Also, there is limited knowledge of its toxic mechanism, including disruption of calcium homeostasis by activation of calcium-dependent kinases, phospholipases, proteases [15], myofibrillar disruption, apoptotic cell death, as well as mitochondrial dysfunction. The mitochondrial toxic effect of DOX relates to the generation of ROS, destroying energy production [44]. The impact on its mitochondrial toxicity is caused by blocking the ETC associated with cardiolipin, which is an inner mitochondrial membrane protein [44]. DOX's toxicity is mainly associated with enhanc-

330 Mitochondrial Diseases

ing mitochondrial ROS production and decreasing mitochondrial biogenesis [38].

a mystery. Therefore, further studies are needed to increase knowledge [21].

However, its clinical utility is limited due to irreversible myocardial damage and dysfunction. Apoptosis mediated by DOX contributes to heart failure. DOX's main intracellular target is mitochondria, causing mitochondrial damage and ROS elevation, and initiating apoptosis [21]. So, DOX gives rise to degrading contractile proteins [29]. However, limited knowledge of how mitochondrial dysfunction triggers cardiac apoptotic cell death-mediated DOX is still

DOX has detrimental effects, classified as acute and chronic abnormalities, including arrhythmias, heart failure, and ventricular dysfunction. The primary issue for DOX therapy is to overcome and minimize its toxic effect without altering its therapeutic impact on cells with cancer. Knowledge of its detrimental effect remains a mystery. There are, however, disorders that may explain its side effect, such as mitochondrial dysfunction and ROS production. Mitochondria have an essential function, including energy metabolism, cellular apoptosis, and cell death pathways, apoptosis, and necrosis [35]. The cardiotoxicity of DOX relies on its dosage. For example, electrocardiologic abnormalities have been reported to occur at a low dose, although dilated cardiomyocytes and congestive heart failure have been reported at a high dose [13]. After left ventricular end diastolic pressure and left ventricular ejection fraction are suppressed, DOX dilates cardiomyopathy because of the decline in heart pump function. Besides cardiomyopathy, DOX also leads to the development of cardiac remodeling, including cytoplasmic vacuolization, myofibrillar clutter, or sarcoplasmic reticulum (SR) swelling. This is why further studies are required to evaluate DOX's toxic effects on noncancerous tissue [4]. There is no defined specific therapy to cope with DOX's cardiomyopathy yet, except receiving traditional treatment of congestive heart failure, e.g., angiotensin converting enzyme blockers, etc. [24].

Based on our best knowledge of the mechanisms associated with apoptosis, oxidative stress, and mitochondrial dysfunction, to avoid undesired toxicity it has been suggested to use some form of antioxidant. Unfortunately, antioxidant therapy has failed to accomplish the drug's toxicity effect in many tissues, particularly the heart and liver according to clinical data [39]. This is why any approaches to use the drug clinically may reduce its toxic effects on noncancerous mass. Therefore, further studies are needed to evaluate the molecular mechanism of DOX's toxicity [45].

The most severe toxic effect of DOX is on the heart [17]. This toxic effect is related to mitochondria because DOX targets cellular mitochondria, resulting in mitochondrial damage and cell death [20]. Cardiomyocytes are differentiated and nondividing cells, so they would not be a direct target of the drug since it blocks DNA replication and synthesis [28]. Therefore, cardiomyocytes have an insufficient regenerative ability after significant injury [46, 47]. In case of severe damage, the majority of heart muscle functions can be terminally lost. DOX could selectively oxidize mtDNA associated with heart failure [28]. For some reasons the most severe detrimental effect of DOX is seen as heart based. These reasons are:


Cardiomyocytes contain high mitochondrial density, and one cardiomyocyte occupies 40–45% of mitochondria [21, 34]. The organelle has a function to maintain standard cardiac capacity due to a high-demanding, high-energy substrate for contractile function [21]. DOX accumulates in mitochondria 100 times more than plasma [34]. After binding DOX, cardiolipin loses the cofactor role in mitochondrial enzymes [34].

DOX tends to accumulate in the nucleus and mitochondria. In heart tissue, mitochondria make up around 50% of its volume [48]. DOX has a high affinity to bind the inner mitochondrial membrane and is collected on the matrix side [3]. One of DOX's similarities in the inner mitochondrial membrane is cardiolipin, which has a much higher affinity vs. other lipids in mitochondria (around 80 times). Phosphatidylethanolamine and cardiolipin are adaptors in the hexagonal (HII) phase in existent divalent cations, e.g., DOX, leading to changes in fluidity and functionality of mitochondrial membranes. DOX inactivates mitochondrial lipiddependent enzymes, such as NADH dehydrogenase, cytochrome-*c* oxidase, and cytochrome*c* reductase. DOX binds to cardiolipin, causing inactivation of complex I–III. DOX and NADH/ NADH dehydrogenase incubations have been suggested to reduce sequestration at the SR by around 80% [48]. Also, mitochondrial TOPI is also found to relate to anthracyline-based cardiac toxicity [11].

The heart's mitochondria have two NADH dehydrogenases. One, known as cytosolic or intermembranous, is located at the outer surface of the inner mitochondrial membrane. However, the other one, known as matrix NADH dehydrogenase, is placed at the matrix surface of the inner mitochondrial membrane. Complex I relates to cytosolic NADH dehydrogenase as a function to capture the electrons from the mitochondrial cytosol to the electron transport system (ETS). Moreover, cytosolic NADH dehydrogenase probably participates in DOX-induced heart toxicity. The molecular weight of DOX is around 600 Da. So, DOX with a hydrophilic structure could smoothly transit from the outer membrane to the mitochondrial cytosol. However, it is difficult to pass through an inner mitochondrial membrane with a lipoidal structure. Therefore, DOX cannot reach the matrix NADH dehydrogenase. This is why DOX is almost impossible to convert its semiquinone form at most cell types, e.g., renal or hepatic tissues and tumor cells as well. On the other hand, heart tissue contains cytosolic NADH dehydrogenase at mitochondria. This is why DOX can be converted to its semiquinone form, leading to oxidative stress by transferring one electron to molecular oxygen [10]. Furthermore, the semiquinone form can produce dihydroquinone via itself by deletion of the sugar moiety to make its aglycone form. The primary metabolites are suggested to be of aglycone form because the form can easily pass through the inner membrane due to its lipoidal structure. In this way, the major form could substitute coenzyme-Q10 and block complex I and II as well at around a 100 μM concentration. Thus, this results in dissociation of coenzyme-Q10 from mitochondria. This is why the plasma coenzyme-Q10 level is increased in cancer patients receiving DOX therapy and decreased in heart tissue as well. The aglycone form of DOX could deliver electrons to an oxygen molecule, enhancing the superoxide radical. Superoxide dismutase at mitochondria can serve to convert hydrogen peroxide (H2 O2 ) to hydroxyl radicals and water, which is why heart tissue is susceptible to oxidative stress produced by ETS and the DOX semiquinone form as well. The other detrimental effect of the aglycone form breaks energy synthesis from mitochondria due to the substitution of coenzyme-Q10 acting as a potent antioxidant. Aglycone derivatives of DOX lose the anticancer impact of the drug because it does not bind to DNA [10].

lower its binding to the membrane. This knowledge is vital when the inner mitochondrial membrane is thought not to contain cholesterol. So, cholesterol and DOX or DOX's derivatives as semiquinone compete with binding of the hydrophobic region of the mitochondrial membrane. There are reasons why mitochondrial lipid peroxidation is high. The first reason is that the outer mitochondrial layer produces more ROS. The second reason is that the inner mitochondrial membrane is a very rich nonsaturated fatty acid. The third reason is that cardiolipin exists as 18% of total lipids in the mitochondria. So, DOX has a very high affinity for cardiolipin [48]. DOX tends to accumulate in mitochondria; therefore, mitochondrial ROS and RNS can be produced [28]. Elevation of ROS causes the enhancement of NF-κB and inducible nitric oxide synthase (iNOS) [28]. This process could trigger a positive feedback. iNOS also

**Figure 3.** The formation of a semiquinone radical (SQ) from doxorubicin (DOX) by capturing one electron, which causes auto-oxidation in existing molecular oxygen, resulting in superoxide radical formation. Modified from Carvalho et al.

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 333

It is well known that DOX's toxicity is based on its cumulative dose. It is reported that DOX could be lethal when mice are treated with DOX as a single dose of 12.5–25 mg/kg or two 15 mg/kg doses. Thus, the survival rate of drug treatment is between 40 and 0% at lower and higher doses, respectively [10]. DOX's toxicity has been classified as acute and chronic. Its acute effect occurs when patients receive drug treatment and has been reported to show transient arrhythmias, hypotension, and pericarditis. However, chronic DOX's results are evident even years after treatment and give rise to more severe damage, including congestive heart

Acute toxicity has been seen by electrocardiographic (ECG) alternation as suppression of myocardial contractile function [10]. Another myocardial dysfunction induced by the acute DOX

initiates to form ROS, which will be looked at in another section of this chapter.

**5.1. The acute toxic effect of doxorubicin**

[17].

failure and dilated cardiomyopathy [43].

Excess electrons generated are captured by oxidizing agents, such as oxygen, and the cardiac tissue has a very high oxygen consumption rate [36]. Heart tissue needs more energy to maintain contractile function and cell survival, which is why cardiomyocytes have substantial mitochondrial volume. The mechanism of DOX's toxicity is still a mystery. However, many studies have suggested the association between ROS and reactive nitrogen species (RNS) with their side effects [20] (**Figure 3**). In other words, the heart has been extensively exposed to oxidative stress. The reason for this is due to the enormous volume of mitochondria and weak antioxidant defense in the tissue [17]. The heart contains low-level catalase enzymes; in addition, DOX immediately inactivates selenium-dependent glutathione (GSH)-peroxidase-1 and cytosolic Cu– Zn superoxide dismutase enzymes after therapy [17, 36, 42]. DOX is claimed to have a univalent redox potential of around −320 mV [17]. This fact can be combined with information that a high proton concentration might have potential to enhance mitochondrial ROS production [49]. Based on this potential, DOX is a suitable substrate for certain oxidoreductase enzymes, which are NADPH-dependent cytochrome P450 reductase, NADH dehydrogenase, and xanthine oxidase. DOX is highly reduced by complex I, resulting in semiquinone. It is well determined to have DOX affinity to cardiolipin with phospholipids. Cardiolipin acts as a cofactor for respiratory chain enzymes, e.g., cytochrome-c oxidase and NADH cytochrome-c oxidoreductase [17].

The semiquinone and molecular oxygen reaction is very fast (*k =* 108 M−<sup>1</sup> s−<sup>1</sup> ). Semiquinone and H2 O2 can be catalyzed under very low oxygen conditions. Cholesterol is a crucial element to determine the localization and/or association of the drug. If cholesterol is high, DOX can

**Figure 3.** The formation of a semiquinone radical (SQ) from doxorubicin (DOX) by capturing one electron, which causes auto-oxidation in existing molecular oxygen, resulting in superoxide radical formation. Modified from Carvalho et al. [17].

lower its binding to the membrane. This knowledge is vital when the inner mitochondrial membrane is thought not to contain cholesterol. So, cholesterol and DOX or DOX's derivatives as semiquinone compete with binding of the hydrophobic region of the mitochondrial membrane. There are reasons why mitochondrial lipid peroxidation is high. The first reason is that the outer mitochondrial layer produces more ROS. The second reason is that the inner mitochondrial membrane is a very rich nonsaturated fatty acid. The third reason is that cardiolipin exists as 18% of total lipids in the mitochondria. So, DOX has a very high affinity for cardiolipin [48]. DOX tends to accumulate in mitochondria; therefore, mitochondrial ROS and RNS can be produced [28]. Elevation of ROS causes the enhancement of NF-κB and inducible nitric oxide synthase (iNOS) [28]. This process could trigger a positive feedback. iNOS also initiates to form ROS, which will be looked at in another section of this chapter.
