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

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

The semiquinone and molecular oxygen reaction is very fast (*k =* 108 M−<sup>1</sup> s−<sup>1</sup>

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

because it does not bind to DNA [10].

332 Mitochondrial Diseases

and H2 O2

cals 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

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].

O2

) to hydroxyl radi-

). Semiquinone

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 failure and dilated cardiomyopathy [43].

Acute toxicity has been seen by electrocardiographic (ECG) alternation as suppression of myocardial contractile function [10]. Another myocardial dysfunction induced by the acute DOX effect is diastolic dysfunction after therapy. Although there are no severe symptoms of diastolic dysfunction, it is becoming a very crucial issue for chronic DOX therapy due to concomitant systolic dysfunction [24]. The signs are transient electrophysiological alternations, including sinus tachycardia, supraventricular, and reversible arrhythmias, ST- and T-wave alternations, prolonged QT interval, QRS voltage decline, and flattening of the T wave, predicted at 11% within all cases [14, 17]. Some symptoms have been reported to appear rarely but are more severe, e.g., pericarditis, myocarditis, and acute left ventricular failure [17]. Also, one of the previous studies indicated that DOX led to pericardial, peritoneal, and pleural effusion [49]. The other severe side effects are hyperpigmentation of the skin veins used for drug injection, stomatitis, and myelosuppression [18]. Moreover, other acute effects cause loss of body, heart, and liver weights and also enhance lipid peroxidation [10]. Acute drug-exposure is suggested to cause ROS generation from complex I at ETC in mitochondria [13], as well as the initiation of apoptosis [50]. With this knowledge, our previous studies have shown that antioxidant supplementation might be an excellent candidate to moderate DOX's toxicity [51–56].

**3.** Bioalkylation at C7 aglycone by metabolic activation, resulting in alkylation and destruc-

**4.** A high affinity for iron (both ferric and ferrous forms) and copper so the drug can reduce

Chronic DOX therapy leads to heart failure; cardiomyopathy has been reported to be associated with oxidative stress and mitochondrial dysfunction [58]. Mitochondria are essential organelles that synthesize ATP with a total of four membrane-associated complexes: complex I, II, III, and IV. The amount of ATP production is related to the complexes' activities (**Figure 3**). For example, high activity produces more ATP, although low activity has the opposite effect. DOX is toxic to mitochondria; all complexes could be inhibited, leading to energy stress. A recent study result showed that cryptotanshinone treatment, which is obtained from *Salvia miltiorrhiza* root, could reverse the toxic compound effect of DOX, except complex II (succinate dehydrogenase) by elevation of MMP, resulting in enhanced ATP formation [58]. How the increase in MMP by cryptotanshinone treatment occurs can be explained by a decline in free radicals, particularly superoxide anion. Since the increase in oxidative stress destroys the reduction/oxidation balance in mitochondria, DOX has been well accepted to elevate ROS generation [58]. So, the cryptotanshinone mitigates the imbalance, eventually increasing both MMP and ATP production [58]. However, lengthy drug exposure is reported not to be related to drug interaction with ETC enzymes, but the molecular mechanism of extended DOX treatment to produce ROS is so far not well understood. So, further studies are required to evalu-

DOX has been reported to cause severe histological and electrophysiological (electrocardiogram) alternations of cardiac tissue related to cardiomyopathy, and also creatine phospho-

gain has been reported in animal research with chronic DOX therapy. Histopathological and electrophysiological, including flattened-inverted T wave and declining QRS voltage, alternations have appeared in chronic DOX therapy [10]. Chronic DOX's toxicity led to more severe arrhythmias, including sudden death [59]. High blood pressure was reported after DOX treatment [60]. Histopathological variation of DOX's cardiotoxicity is observed as myofibrillar loss, sarcoplasmic swelling, cytoplasmic, myelin, and mitochondrial vacuolization, and crystal degeneration in mitochondria [23]. The acute and chronic toxic effects of DOX are

Under the standard physiologic condition, ROS can be by synthesis only 1–5% of oxygen consumption [11]. The most acceptable hypothesis of DOX's toxicity is extensive ROS production [4]. The reason for elevation by DOX is associated with its accepting and donating electrons. DOX contains a hexose sugar with tetracycline having quinone and hydroquinone moieties, which are part of the capture electron, producing semiquinone. A superoxide radical can be provided by semiquinone from an oxygen molecule. A superoxide radical does not have a

cumulative dose. In contrast to acute studies, body weight

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 335

activation through metal chelating effects, leading to free radical formation [48].

ate the production of ROS induced by lengthy DOX treatment [13].

**5.3. The mechanism of reactive oxygen species production of doxorubicin**

tion of DNA [48].

kinase elevation at 450 mg/m2

summarized in **Table 1**.

Interestingly, a transient DOX effect has been reported to shift mitochondrial dynamics to fission at the heart. However, acute DOX therapy at the liver tissue is reported to decrease fusion, but not alter fission. This means that a decrease in fusion leads to an increase in mitochondrial fragmentation in the liver. DOX is said to improve mitophagy at the organ. When mitochondrial fusion and mitophagy occur, mitochondrial content reduces. DOX also causes a decrease in citrate synthase. Acute DOX has been mentioned not to change proliferating cell nuclear antigen, which means that mitochondrial fission does not accompany cell proliferation [39].

However, all acute toxic effects are transient, occur within the first 24 h after drug therapy, and are spontaneously ameliorated [10]. Sometimes an acute DOX effect can transiently appear and disappear within a few minutes to a week. Acute DOX is prevalent in cancer patients receiving DOX therapy at around 20–30% [17]. The chronic toxic effects of DOX cause cardiomyopathy and congestive heart failure [10].

#### **5.2. The chronic toxic effect of doxorubicin**

The chronic toxic effect of DOX results in an irreversible defect in cardiomyopathy, congestive heart failure. A dose of DOX at 430–600 mg/m2 given to 50–60% of patients has been reported to develop left ventricular failure. However, a cumulative dose of DOX at 300 mg/m2 has been shown to increase heart failure by almost 2%. Moreover, causing heart failure induced by DOX is quickly enhanced after a 550 mg/m2 dose [17], which is a limited dosage because it induces irreversible toxicity [48]. To see the chronic effects of the drug takes a year of therapy. However, rapid treatment still leads to damaged heart tissue [17]. Other toxicities of DOX have been reported to be palmar–plantar erythrodysesthesia (also known as a hand–foot syndrome) [48] and typhlitis [57]. DOX's toxic impact on tissue is associated with:


**3.** Bioalkylation at C7 aglycone by metabolic activation, resulting in alkylation and destruction of DNA [48].

effect is diastolic dysfunction after therapy. Although there are no severe symptoms of diastolic dysfunction, it is becoming a very crucial issue for chronic DOX therapy due to concomitant systolic dysfunction [24]. The signs are transient electrophysiological alternations, including sinus tachycardia, supraventricular, and reversible arrhythmias, ST- and T-wave alternations, prolonged QT interval, QRS voltage decline, and flattening of the T wave, predicted at 11% within all cases [14, 17]. Some symptoms have been reported to appear rarely but are more severe, e.g., pericarditis, myocarditis, and acute left ventricular failure [17]. Also, one of the previous studies indicated that DOX led to pericardial, peritoneal, and pleural effusion [49]. The other severe side effects are hyperpigmentation of the skin veins used for drug injection, stomatitis, and myelosuppression [18]. Moreover, other acute effects cause loss of body, heart, and liver weights and also enhance lipid peroxidation [10]. Acute drug-exposure is suggested to cause ROS generation from complex I at ETC in mitochondria [13], as well as the initiation of apoptosis [50]. With this knowledge, our previous studies have shown that antioxidant

supplementation might be an excellent candidate to moderate DOX's toxicity [51–56].

Interestingly, a transient DOX effect has been reported to shift mitochondrial dynamics to fission at the heart. However, acute DOX therapy at the liver tissue is reported to decrease fusion, but not alter fission. This means that a decrease in fusion leads to an increase in mitochondrial fragmentation in the liver. DOX is said to improve mitophagy at the organ. When mitochondrial fusion and mitophagy occur, mitochondrial content reduces. DOX also causes a decrease in citrate synthase. Acute DOX has been mentioned not to change proliferating cell nuclear antigen, which means that mitochondrial fission does not accompany cell proliferation [39].

However, all acute toxic effects are transient, occur within the first 24 h after drug therapy, and are spontaneously ameliorated [10]. Sometimes an acute DOX effect can transiently appear and disappear within a few minutes to a week. Acute DOX is prevalent in cancer patients receiving DOX therapy at around 20–30% [17]. The chronic toxic effects of DOX cause

The chronic toxic effect of DOX results in an irreversible defect in cardiomyopathy, congestive

been shown to increase heart failure by almost 2%. Moreover, causing heart failure induced

induces irreversible toxicity [48]. To see the chronic effects of the drug takes a year of therapy. However, rapid treatment still leads to damaged heart tissue [17]. Other toxicities of DOX have been reported to be palmar–plantar erythrodysesthesia (also known as a hand–foot syn-

**1.** A high affinity to bind membrane lipid-dependent pH, resulting in membrane alternation

to develop left ventricular failure. However, a cumulative dose of DOX at 300 mg/m2

drome) [48] and typhlitis [57]. DOX's toxic impact on tissue is associated with:

given to 50–60% of patients has been reported

dose [17], which is a limited dosage because it

has

cardiomyopathy and congestive heart failure [10].

**5.2. The chronic toxic effect of doxorubicin**

334 Mitochondrial Diseases

heart failure. A dose of DOX at 430–600 mg/m2

by DOX is quickly enhanced after a 550 mg/m2

of lipid structure by lipid peroxidation [48].

**2.** Production of a semiquione structure [48].

**4.** A high affinity for iron (both ferric and ferrous forms) and copper so the drug can reduce activation through metal chelating effects, leading to free radical formation [48].

Chronic DOX therapy leads to heart failure; cardiomyopathy has been reported to be associated with oxidative stress and mitochondrial dysfunction [58]. Mitochondria are essential organelles that synthesize ATP with a total of four membrane-associated complexes: complex I, II, III, and IV. The amount of ATP production is related to the complexes' activities (**Figure 3**). For example, high activity produces more ATP, although low activity has the opposite effect. DOX is toxic to mitochondria; all complexes could be inhibited, leading to energy stress. A recent study result showed that cryptotanshinone treatment, which is obtained from *Salvia miltiorrhiza* root, could reverse the toxic compound effect of DOX, except complex II (succinate dehydrogenase) by elevation of MMP, resulting in enhanced ATP formation [58]. How the increase in MMP by cryptotanshinone treatment occurs can be explained by a decline in free radicals, particularly superoxide anion. Since the increase in oxidative stress destroys the reduction/oxidation balance in mitochondria, DOX has been well accepted to elevate ROS generation [58]. So, the cryptotanshinone mitigates the imbalance, eventually increasing both MMP and ATP production [58]. However, lengthy drug exposure is reported not to be related to drug interaction with ETC enzymes, but the molecular mechanism of extended DOX treatment to produce ROS is so far not well understood. So, further studies are required to evaluate the production of ROS induced by lengthy DOX treatment [13].

DOX has been reported to cause severe histological and electrophysiological (electrocardiogram) alternations of cardiac tissue related to cardiomyopathy, and also creatine phosphokinase elevation at 450 mg/m2 cumulative dose. In contrast to acute studies, body weight gain has been reported in animal research with chronic DOX therapy. Histopathological and electrophysiological, including flattened-inverted T wave and declining QRS voltage, alternations have appeared in chronic DOX therapy [10]. Chronic DOX's toxicity led to more severe arrhythmias, including sudden death [59]. High blood pressure was reported after DOX treatment [60]. Histopathological variation of DOX's cardiotoxicity is observed as myofibrillar loss, sarcoplasmic swelling, cytoplasmic, myelin, and mitochondrial vacuolization, and crystal degeneration in mitochondria [23]. The acute and chronic toxic effects of DOX are summarized in **Table 1**.
