**3. Anticancer drugs-induced tissue injury: The role of mitochondrial dysfunction**

Mitochondria are dynamic and multifunctional cytoplasmic organelles, which possess a double membrane: an outer membrane (OMM) that is essentially permeable to ions and solutes up to 14 kDa, and an inner membrane (IMM), which is folded, forming the cristae, which is impermeable to ions and polar molecules. In the IMM are located several transporters, including the ATP/ADP and the aspartate/malate transporters, among others that regulate the movements of molecules across the IMM, and also the multisubunit complexes involved in the oxidative phosphorylation. Between the OMM and the IMM is located the intermembrane space (IMS), whereas the space enclosed by the IMM is the matrix (Fig.1). The IMS contains proteins such as the adenylate kinase and the creatine kinase (CK), as well as the cytochrome *c* and the apoptosis-inducing factor (AIF), which translocate to the cytoplasm during the apoptotic process, and other proteins involved in cellular metabolism. The proteins involved in the Krebs cycle, in the fatty acid oxidation, as well as in the synthesis of the heme and steroids, are located in the matrix [8, 9, 12, 13]. The mitochondrial matrix also contains the mitochondrial DNA (mtDNA). Thus, mitochondria are not only responsible for the synthesis of most of the ATP produced in the cell, but they also play a role in the fatty acid oxidation, the synthesis of the heme, steroids, and polypeptides involved in energy generation, as well as in calcium regulation and cell death. Therefore, taken in account the multitude of essential functions of mitochondria in cells, it is expected that their dysfunction might promote tissue failure, as described below.

#### **3.1. Disturbance of mitochondrial energy production**

The NADH and FADH2 generated in metabolic pathways, including glycolysis, fatty acid oxidation and Krebs cycle are oxidized by complexes I and II of the mitochondrial electron transport chain, respectively. The electrons liberated are then passed to ubiquinone (coenzyme Q) that shuttles electrons to complex III, where it is oxidized; the electrons are then passed to complex IV through cytochrome *c*. The electrons carried by cytochrome *c* are used by complex IV to reduce molecular oxygen to water (Fig.1) [8, 14]. According to Peter Mitchell's Chemiosmotic Theory, complexes I, III and IV are redox-driven proton pumps that harness the energy derived from oxidation-reduction reactions to pump protons into the IMS, in parallel to the electron transfer, creating the electrochemical proton gradient (proton motive force) (ΔµH<sup>+</sup> ), which is comprised of two components: a pH gradient (ΔpH) and a membrane potential (ΔΨ) (Fig. 1). The complex V (ATP synthase), which consists of the F1 subunit, a soluble portion located in the mitochondrial matrix, and the Fo subunit, bound to the IMM, uses the electrical component ΔΨ to phosphorylate ADP to ATP. Thus, the oxidative phosphorylation is coupled to the ATP requirements and the electron flow along the electron transport chain only occurs when the synthesis of ATP is required. The IMM impermeability ensures that the proton pumping along the respiratory chain is coupled to ATP synthesis [8, 12, 14].

**Figure 1.** Structure of mitochondria and main mechanisms by which anticancer agents affect mitochondrial functions. Drugs can compromise mitochondrial bioenergetic functions by interfering with the generation system of ΔµH<sup>+</sup> , by dissipating the transmembrane proton gradient and by directly affecting the complex V or the substrate transporters. Drugs can also trigger or inhibit the mitochondrial permeability transition (MPT) by interfering with the proteins that compose or regulate the MPT or by modulating the critical factors to its onset, which may ultimately lead to cell death. Other drugs induce changes in the redox regulation of mitochondrial functions, promoting several deleterious events, including the interference with oxidative phosphorylation, nutrient oxidation and MPT. Drugs can also damage the mtDNA and, indirectly, compromise the ATP synthesis and favor the production of ROS (ANT, adenine nucleotide translocase; CyPD, cyclophilin-D; Cytc, cytochrome *c*; mnSOD, manganese superoxide dismutase; mtDNA, mitochon‐ drial DNA; PiC, phosphate carrier; Prx, glutathione peroxidase; Q, coenzyme Q).

Xenobiotics can affect mitochondrial bioenergetic functions either by interfering with the generation system of ΔµH<sup>+</sup> or by causing the dissipation of the transmembrane proton gradient, as well as by affecting directly the F1Fo ATPase and the substrate transporters, including the adenine nucleotide translocase (ANT) and the phosphate–hydrogen co-transporter (phos‐ phate carrier) (Fig.1) [14].

In fact, the inhibition of the electron transport chain by drugs may not only lead to ATP depletion, but also hinders the reoxidation of NADH and FADH2 into NAD<sup>+</sup> and FAD, respectively, which are required for the activity of several dehydrogenases of the Krebs cycle and the mitochondrial β-oxidation [15, 16]. In addition, the inhibition of the electron transport chain favors the accumulation of electrons in the electron transport system complexes that can escape and directly react with oxygen to form the superoxide anion radical [17]. The excessive reactive oxygen species (ROS) production can promote several deleterious events (described in more detail in section 3.3), but it is noteworthy that, in this context, both lipid accumulation and lipid peroxidation are favored [15, 16].

On the other hand, drugs can compromise the impermeability of the IMM to protons and dissipate the proton gradient impairing the production of ATP. The IMM impermeability can be surpassed by drugs that either disrupt the mitochondrial membranes or act as ionophores, uncouplers of the oxidative phosphorylation and inducers of the mitochondrial permeability transition (MPT) [12-14]. Moreover, drugs that interfere with the basic components of the phosphorylative system can also impair the production of ATP. These different effects on mitochondria induce changes in cellular bioenergetics, one of the key hallmarks in tissues.

#### **3.2. Mitochondria in Ca2+homeostasis: MPT-dependent cell death**

and a membrane potential (ΔΨ) (Fig. 1). The complex V (ATP synthase), which consists of the F1 subunit, a soluble portion located in the mitochondrial matrix, and the Fo subunit, bound to the IMM, uses the electrical component ΔΨ to phosphorylate ADP to ATP. Thus, the oxidative phosphorylation is coupled to the ATP requirements and the electron flow along the electron transport chain only occurs when the synthesis of ATP is required. The IMM impermeability ensures that the proton pumping along the respiratory chain is

**Figure 1.** Structure of mitochondria and main mechanisms by which anticancer agents affect mitochondrial functions. Drugs can compromise mitochondrial bioenergetic functions by interfering with the generation system of ΔµH<sup>+</sup>

dissipating the transmembrane proton gradient and by directly affecting the complex V or the substrate transporters. Drugs can also trigger or inhibit the mitochondrial permeability transition (MPT) by interfering with the proteins that compose or regulate the MPT or by modulating the critical factors to its onset, which may ultimately lead to cell death. Other drugs induce changes in the redox regulation of mitochondrial functions, promoting several deleterious events, including the interference with oxidative phosphorylation, nutrient oxidation and MPT. Drugs can also damage the mtDNA and, indirectly, compromise the ATP synthesis and favor the production of ROS (ANT, adenine nucleotide translocase; CyPD, cyclophilin-D; Cytc, cytochrome *c*; mnSOD, manganese superoxide dismutase; mtDNA, mitochon‐

Xenobiotics can affect mitochondrial bioenergetic functions either by interfering with the

as well as by affecting directly the F1Fo ATPase and the substrate transporters, including the adenine nucleotide translocase (ANT) and the phosphate–hydrogen co-transporter (phos‐

In fact, the inhibition of the electron transport chain by drugs may not only lead to ATP

respectively, which are required for the activity of several dehydrogenases of the Krebs cycle

depletion, but also hinders the reoxidation of NADH and FADH2 into NAD<sup>+</sup>

or by causing the dissipation of the transmembrane proton gradient,

drial DNA; PiC, phosphate carrier; Prx, glutathione peroxidase; Q, coenzyme Q).

generation system of ΔµH<sup>+</sup>

phate carrier) (Fig.1) [14].

, by

and FAD,

coupled to ATP synthesis [8, 12, 14].

26 Toxicology Studies - Cells, Drugs and Environment

Besides serving as the cells' primary energy source, mitochondria are implicated in the maintenance of calcium homeostasis through calcium uptake and release pathways. The rise of mitochondrial matrix free calcium concentration in the presence of a variety of sensitizing factors can lead to the opening of the MPT pore that may serve either the purpose of providing a fast calcium release or can convey both apoptotic and necrotic death signals.

MPT can be defined as an increase in the IMM permeability to solutes with molecular masses up to 1 500 Da, due to the opening of a voltage and calcium-dependent, cyclosporine A (CyA) sensitive channel [18].

The molecular identity of the MPT pore is still under debate [19] (Fig. 1). Cyclophilin-D is the binding site for CyA, the golden standard of MPT inhibitors, and among the several compo‐ nents that have been proposed to play a role on MPT, cyclophilin-D is probably the most consensual. The ANT and the voltage-dependent anion channel (VDAC) were also considered key components of the MPT pore complex for many years. Knockout mice devoid of cyclo‐ philin-D are resistant to necrosis promoted by ROS and calcium overload and the mitochondria isolated from the liver, heart and brain of these animals are resistant to MPT *in vitro* [20, 21]. Although these results support a central role for cyclophilin-D, MPT can occur even in the absence of cyclophilin-D if the calcium concentration is high enough [22]. Likewise, MPT can occur in mitochondria lacking ANT, although a higher concentration of calcium is required [23] and, therefore, the ANT is now considered a regulatory component of the MPT pore, rather than a structural one. Similar studies have excluded the VDAC as an essential component of MPT pore megacomplex [24].

The phosphate carrier was also proposed to play a key role in MPT [25], but a later study demonstrated that the reduction of phosphate carrier protein expression does not affect the MPT [26]. A more recent work suggested that the MPT pore complex is composed of ATP synthase dimers and pointed out that cyclophilin-D binds the lateral stalk of the ATP synthase at the same site as benzodiazepine 423, increasing the sensitivity to calcium [27]. However, the composition of MPT still remains a contentious issue.

Besides the calcium concentration in the matrix, the level of oxidative stress is possibly the most critical factor regulating MPT pore opening. Indeed, both the ANT and cyclophilin D were shown to be modulated by S-oxidation reactions [28-29]. However, other factors may contribute as well: the depletion of adenine nucleotides and high concentrations of phosphate increase the sensitivity of MPT to calcium, while a low pH and a high (negative) ΔΨ inhibit MPT pore opening [30].

Drugs can trigger MPT either by interfering with the proteins that compose or regulate the MPT or by modulating the critical factors to its onset (Fig.1) [8]. As a consequence of MPT pore opening, the IMM loses its impermeability to protons, which allows the movement of solutes between the matrix and the cytosol, the dysregulation of cellular ionic homeostasis and the dissipation of ΔµH<sup>+</sup> . Moreover, since the protein concentration is higher in the matrix, a high osmotic pressure is generated and may lead to mitochondrial swelling, rupture of OMM, and loss of proapoptotic proteins that may trigger the apoptotic pathway in the cytoplasm [30, 31]. These events, together with the bioenergetic failure and the redox catastrophe, can culminate in cell death; if there are no sufficient levels of ATP, necrotic death may predominate over apoptosis [32]. Thus, depending on the cell type involved, different pathological condi‐ tions can occur as consequence of MPT induction.

#### **3.3. Changes in redox regulation of mitochondrial functions**

During the transfer of electrons along the electron transport chain to oxygen, and particularly at complexes I and III, some of these electrons escape and directly react with oxygen. The univalent reduction of oxygen generates the superoxide anion radical, which is then dismu‐ tated by the mitochondrial manganese superoxide dismutase into hydrogen peroxide, a key ROS signaling molecule due to its longer half-life and capacity to diffuse through membranes. The hydrogen peroxide is detoxified into water by the mitochondrial glutathione peroxidase and hence, in normal circumstances, most of the ROS generated by the electron transport chain are neutralized by the mitochondrial antioxidant defenses (Fig.1) [17]. The accumulation of hydrogen peroxide can promote the oxidation of thiolic groups, irreversibly deactivating the protein; likewise, the superoxide anion disassembles Fe–S clusters in several Krebs cycle enzymes and in respiratory complexes, and can combine with nitric oxide to form the highly toxic peroxynitrite. In addition, high levels of ROS can lead to the production of hydroxyl radical, which indiscriminately oxidizes biological macromolecules [33]. Other sources that can contribute to the total mitochondrial ROS include 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase, dihydroorotate dehydrogenase, sn-glycerol-3-phosphate dehydrogenase, electron transfer flavoprotein:ubiquinol oxidoreductase, p66shc/Cytochrome C, Mia40p/ Erv1p, and complex II [33]. When maintained within a certain concentration range, ROS act as important signaling molecules, contributing to the redox balance, which regulates the functions that assist the normal cellular physiology [33]. However, the excessive formation of ROS can promote a series of deleterious events that deregulate key mitochondrial functions, including oxidative phosphorylation, nutrient oxidation and MPT, which may ultimately lead to cell death, tissue failure and have also a key role in disease pathogenesis [33].

#### **3.4. Mitochondrial DNA damage**

at the same site as benzodiazepine 423, increasing the sensitivity to calcium [27]. However, the

Besides the calcium concentration in the matrix, the level of oxidative stress is possibly the most critical factor regulating MPT pore opening. Indeed, both the ANT and cyclophilin D were shown to be modulated by S-oxidation reactions [28-29]. However, other factors may contribute as well: the depletion of adenine nucleotides and high concentrations of phosphate increase the sensitivity of MPT to calcium, while a low pH and a high (negative) ΔΨ inhibit

Drugs can trigger MPT either by interfering with the proteins that compose or regulate the MPT or by modulating the critical factors to its onset (Fig.1) [8]. As a consequence of MPT pore opening, the IMM loses its impermeability to protons, which allows the movement of solutes between the matrix and the cytosol, the dysregulation of cellular ionic homeostasis and the

osmotic pressure is generated and may lead to mitochondrial swelling, rupture of OMM, and loss of proapoptotic proteins that may trigger the apoptotic pathway in the cytoplasm [30, 31]. These events, together with the bioenergetic failure and the redox catastrophe, can culminate in cell death; if there are no sufficient levels of ATP, necrotic death may predominate over apoptosis [32]. Thus, depending on the cell type involved, different pathological condi‐

During the transfer of electrons along the electron transport chain to oxygen, and particularly at complexes I and III, some of these electrons escape and directly react with oxygen. The univalent reduction of oxygen generates the superoxide anion radical, which is then dismu‐ tated by the mitochondrial manganese superoxide dismutase into hydrogen peroxide, a key ROS signaling molecule due to its longer half-life and capacity to diffuse through membranes. The hydrogen peroxide is detoxified into water by the mitochondrial glutathione peroxidase and hence, in normal circumstances, most of the ROS generated by the electron transport chain are neutralized by the mitochondrial antioxidant defenses (Fig.1) [17]. The accumulation of hydrogen peroxide can promote the oxidation of thiolic groups, irreversibly deactivating the protein; likewise, the superoxide anion disassembles Fe–S clusters in several Krebs cycle enzymes and in respiratory complexes, and can combine with nitric oxide to form the highly toxic peroxynitrite. In addition, high levels of ROS can lead to the production of hydroxyl radical, which indiscriminately oxidizes biological macromolecules [33]. Other sources that can contribute to the total mitochondrial ROS include 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase, dihydroorotate dehydrogenase, sn-glycerol-3-phosphate dehydrogenase, electron transfer flavoprotein:ubiquinol oxidoreductase, p66shc/Cytochrome C, Mia40p/ Erv1p, and complex II [33]. When maintained within a certain concentration range, ROS act as important signaling molecules, contributing to the redox balance, which regulates the functions that assist the normal cellular physiology [33]. However, the excessive formation of ROS can promote a series of deleterious events that deregulate key mitochondrial functions,

. Moreover, since the protein concentration is higher in the matrix, a high

composition of MPT still remains a contentious issue.

28 Toxicology Studies - Cells, Drugs and Environment

tions can occur as consequence of MPT induction.

**3.3. Changes in redox regulation of mitochondrial functions**

MPT pore opening [30].

dissipation of ΔµH<sup>+</sup>

The mtDNA, maternally inherited, encodes 13 polypeptides that are subunits of the complexes I, III, IV and V, which are synthesized in mitochondrial ribosomes. The majority of the other mitochondrial proteins, including the subunits of complex II, are encoded by nuclear DNA, and imported into mitochondria after synthesis on cytosolic ribosomes [8-10].

The mtDNA is particularly vulnerable to the action of drugs, as it lacks histones, similarly to the bacterial DNA. Furthermore, the DNA repair mechanisms are less efficient that those of nuclear DNA. Therefore, and considering the proximity to the sites where ROS are routinely generated, the frequency of mtDNA mutation is much higher than that of nuclear DNA [13]. Mutations of mtDNA can seriously damage the respiratory chain as most of the polypeptides that form the respiratory chain complexes are encoded by mtDNA. As discussed in section 3.1., the impairment of the respiratory chain decreases the ATP synthesis capacity and enhances the production of ROS, which in turn will promote oxidative damage on several biomolecules, including the mtDNA itself, creating a vicious cycle that further enhances the insult [34].
