**4. Anticancer drugs impair mitochondria functions: relevance to tissue failure**

In the last decades a large number of anticancer drugs have been reported to induce tissue failure by promoting changes in essential functions of mitochondria. The extent and type of mechanisms underlying the anticancer drug-induced mitochondrial dysfunctions are tissuedependent and responsible for most of the idiosyncratic adverse drug responses.

In this section, we discuss some examples of the prominent members of different groups of anticancer drugs with evidences for the involvement of mitochondrial dysfunction in tissue impairment induced by these compounds.

#### **4.1. Selective estrogen receptor modulators**

Tamoxifen has been the endocrine therapy of choice for women with estrogen-receptor positive breast carcinoma over the last decades. Fatty liver is observed in more than 30 % of patients taking tamoxifen, which may persist after the discontinuation of the treatment [35, 36]. Nonalcoholic steatohepatitis, hepatic fibrosis, cirrhosis and hepatic necrosis were also reported [37-39].

Tamoxifen depresses the phosphorylation efficiency and the levels of ATP in a concentrationdependent manner in isolated rat liver mitochondria; these effects were attributed to a decrease in the active ANT content and a partial inhibition of the phosphate carrier [40, 41]. On the other hand, tamoxifen uncouples liver mitochondria respiration [40, 42] and, at higher concentra‐ tions, tamoxifen disrupts membrane integrity [43-44], enhancing the proton leak [40]. Tamox‐ ifen also inhibits the electron transfer along the electron transport chain [40, 42] and the flavin mononucleotide site of complex I was identified as the target of tamoxifen [45]. The interaction of tamoxifen with complex III and IV was also shown [42]. The fact that tamoxifen interferes with membrane dynamics [46] may also decrease the diffusional mobility of membrane proteins and the electron transfer along the electron transport chain [40]. Furthermore, an *in vivo* study pointed out that tamoxifen depletes hepatic mtDNA, which further contributes to inhibit mitochondrial electron transport chain activity, and triggers steatose in mouse liver [47]. Therefore, the effects promoted by tamoxifen on mitochondrial bioenergetics may contribute to the liver damage observed in patients taking tamoxifen (Fig.1).

Interestingly, tamoxifen active metabolites, 4-hydroxytamoxifen and endoxifen, which are responsible for the antitumor actions of tamoxifen [48], do not significantly compromise mitochondrial bioenergetics at the concentrations reached in tissues [49, 50]. These results may indicate that the clinical use of tamoxifen metabolites instead of the prodrug may minimize liver damage. As the outcome of tamoxifen treatment seems to rely on its metabolic activation and endoxifen is a promising drug for cancer treatment, the future utilization of tamoxifen metabolites, and especially endoxifen, deserves further investigation. On the other hand, tamoxifen prevents and reverses the MPT induced by several agents [51-55]. Likewise, tamoxifen metabolites 4-hydroxytamoxifen and endoxifen also prevent and reverse the MPT induced by calcium and phosphate [50, 56]. Although the mechanisms underlying the inhibition of MPT by the antiestrogens are still under debate [57], this protective effect of TAM and its active metabolites regarding MPT might be of interest when considering combined anticancer drug therapies since it can decrease the toxicity of the associated drugs, as discussed in section 5.

#### **4.2. Antiandrogens**

Flutamide is a nonsteroidal anti-androgen used in the treatment of advanced prostate cancer, which has been associated with idiosyncratic drug-induced liver injury [58] and, although the mechanisms underlying liver damage are still unknown, mitochondria are a potential target of flutamide.

Indeed, high-doses of flutamide promote hepatocytes death in heterozygous Sod2(+/-) mice, but not in wild-type animals, suggesting that flutamide may exacerbate underlying mitochon‐ drial abnormality [59]. Flutamide leads to the covalent binding of reactive electrophilic metabolites to proteins and diminishes the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, as well as the total protein thiols in isolated rat hepatocytes; these effects are associated with the release of lactate dehydrogenase (LDH) [60]. Similar findings were reported by others, which also demonstrated that flutamide increases the hepatic GSSG/GSH ratio, the protein carbonyl levels, and serum lactate levels, supporting the view that the liver damage promoted by flutamide involves oxidative stress and mitochondrondrial dysfunction [59]. Accordingly, the addition of cysteine increased hepatocellular GSH and decreased LDH release in male hepatocytes [60].

Additionally, flutamide markedly impairs rat liver mitochondrial respiration (mainly at the level of complex I) and decreases the levels of ATP in rat hepatocytes [60]. Moreover, flutamidetreated Sod2(+/-) mice present a decrease in the expression of complexes I and III subunits [59]. Therefore, it seems possible that the increased oxidative stress promoted by flutamide may damage mitochondrial proteins and mtDNA, particularly when the antioxidant system is compromised [59], contributing to liver damage (Fig.1).

#### **4.3. Alkylating agents**

hand, tamoxifen uncouples liver mitochondria respiration [40, 42] and, at higher concentra‐ tions, tamoxifen disrupts membrane integrity [43-44], enhancing the proton leak [40]. Tamox‐ ifen also inhibits the electron transfer along the electron transport chain [40, 42] and the flavin mononucleotide site of complex I was identified as the target of tamoxifen [45]. The interaction of tamoxifen with complex III and IV was also shown [42]. The fact that tamoxifen interferes with membrane dynamics [46] may also decrease the diffusional mobility of membrane proteins and the electron transfer along the electron transport chain [40]. Furthermore, an *in vivo* study pointed out that tamoxifen depletes hepatic mtDNA, which further contributes to inhibit mitochondrial electron transport chain activity, and triggers steatose in mouse liver [47]. Therefore, the effects promoted by tamoxifen on mitochondrial bioenergetics may

Interestingly, tamoxifen active metabolites, 4-hydroxytamoxifen and endoxifen, which are responsible for the antitumor actions of tamoxifen [48], do not significantly compromise mitochondrial bioenergetics at the concentrations reached in tissues [49, 50]. These results may indicate that the clinical use of tamoxifen metabolites instead of the prodrug may minimize liver damage. As the outcome of tamoxifen treatment seems to rely on its metabolic activation and endoxifen is a promising drug for cancer treatment, the future utilization of tamoxifen metabolites, and especially endoxifen, deserves further investigation. On the other hand, tamoxifen prevents and reverses the MPT induced by several agents [51-55]. Likewise, tamoxifen metabolites 4-hydroxytamoxifen and endoxifen also prevent and reverse the MPT induced by calcium and phosphate [50, 56]. Although the mechanisms underlying the inhibition of MPT by the antiestrogens are still under debate [57], this protective effect of TAM and its active metabolites regarding MPT might be of interest when considering combined anticancer drug therapies since it can decrease the toxicity of the associated drugs, as discussed

Flutamide is a nonsteroidal anti-androgen used in the treatment of advanced prostate cancer, which has been associated with idiosyncratic drug-induced liver injury [58] and, although the mechanisms underlying liver damage are still unknown, mitochondria are a potential target

Indeed, high-doses of flutamide promote hepatocytes death in heterozygous Sod2(+/-) mice, but not in wild-type animals, suggesting that flutamide may exacerbate underlying mitochon‐ drial abnormality [59]. Flutamide leads to the covalent binding of reactive electrophilic metabolites to proteins and diminishes the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, as well as the total protein thiols in isolated rat hepatocytes; these effects are associated with the release of lactate dehydrogenase (LDH) [60]. Similar findings were reported by others, which also demonstrated that flutamide increases the hepatic GSSG/GSH ratio, the protein carbonyl levels, and serum lactate levels, supporting the view that the liver damage promoted by flutamide involves oxidative stress and mitochondrondrial dysfunction [59]. Accordingly, the addition of cysteine increased hepatocellular GSH and decreased LDH

contribute to the liver damage observed in patients taking tamoxifen (Fig.1).

in section 5.

of flutamide.

**4.2. Antiandrogens**

30 Toxicology Studies - Cells, Drugs and Environment

release in male hepatocytes [60].

#### *4.3.1. Platinum analogs*

Cisplatin, which crosslinks DNA, is widely used in the treatment of head, neck, bladder ovarian and testicular cancers, but it has also been used in the management of other malig‐ nancies. Unfortunately, cisplatin promotes severe side effects, and particularly nephrotoxicity [61] and neurotoxicity [62].

Peripheral neuropathy is the dose limiting side effect of cisplatin and occurs in 30 % of patients. Dorsal root ganglion neurons treated with cisplatin exhibit mitochondrial vacuolization and degradation both *in vitro* and *in vivo*, suggesting that mitochondrial damage is involved in cisplatin-induced neurotoxicity (Fig.1) [63]. Besides covalently binding to nuclear DNA [64], cisplatin also directly binds to mtDNA, hindering the transcription of mitochondrial genes [63]. Another platinum analog, oxaliplatin, affects both complex I- and complex II-mediated respiration and decreases ATP production in rat peripheral nerve axons [65]. Acetyl-lcarnitine, which inhibits the development of oxaliplatin-evoked neuropathy, prevents oxaliplatin-induced mitochondrial dysfunction, further implicating mitochondria in the etiology of peripheral neuropathy [65].

About a quarter to one third of patients undergoing cisplatin treatment experience nephro‐ toxicity, which is manifested clinically as lower glomerular filtration rate and reduced serum magnesium and potassium levels [61]. Although the mechanism underlying cisplatin neph‐ rotoxicity is not yet clear, the available evidence suggests that mitochondrial dysfunction plays a key role in renal tubular cell injury and death. Ultrastructural analysis of cisplatin-treated renal tubular cells of mouse kidney demonstrates the decrease in mitochondrial mass, disruption of cristae, and extensive mitochondrial swelling [66], supporting the involvement of mitochondria in cisplatin-induced nephrotoxicity. Cisplatin decreases manganese super‐ oxide dismutase and complex II activity in rodents' kidney [67]. The GSH-reductase activity and the levels of GSH are markedly diminished in porcine proximal tubular cells [68]. These observations suggest that cisplatin strongly reduces the antioxidant defenses and favors ROS formation. However, agents that are able to prevent ROS formation, do not prevent cell death, suggesting that ROS formation is not the direct cause of cell death [68]. Furthermore, cisplatin significantly impairs kidney mitochondrial bioenergetic functions. In porcine proximal tubular cells, cisplatin inhibits complexes I to IV and decreases intracellular ATP [68]. In fact, the kidney of animals treated with cisplatin presents decreased mtDNA content and reduced complex I, III and IV protein expression [67].

Cisplatin is a rare cause of hepatotoxicity (steatosis and cholestasis) at standard doses, but high doses may lead to liver damage, revealed by abnormal liver tests, especially aspartate amino‐ transferase (AST) and alanine aminotransferase (ALT) [69]. Light microscopic observations confirmed that high doses of cisplatin cause massive hepatotoxicity; alterations at the ultra‐ structural level, including atrophied mitochondria, were also found [70]. Cisplatin induces MPT in rat liver mitochondria [71]. Moreover, cisplatin stimulates state 4 respiration, but does not affect FCCP-uncoupled respiration, suggesting that cisplatin affects mitochondrial bioenergetics by increasing the IMM permeability to protons and not by interfering with respiratory chain complexes activity [71]. Both the induction of MPT and the effects on liver mitochondrial bioenergetics are prevented by thiol group protecting agents, suggesting that changes on the redox-status of thiol groups affect membrane permeability to cations and underlie liver mitochondrial dysfunction [71]. Accordingly, it was proposed that the mecha‐ nism of cisplatin-induced hepatotoxicity involves membrane rigidification, lipid peroxidation, oxidative damage of cardiolipin and protein sulfhydryl groups, as well as decreased GSH/ GSSG ratio, ATP, GSH and NADPH [72].

#### *4.3.2. Nitrogen mustards*

Cyclophosphamide is an alkylating agent used in the treatment of lymphomas, multiple myeloma, and certain types of leukemia, retinoblastoma, neuroblastoma, ovarian cancer, and breast cancer. Generally, cyclophosphamide does not cause relevant cardiotoxicity, but when it occurs, it appears to be related to a single dose, unlike the anthracyclines [3]. Patients who were previously medicated with anthracyclines or that underwent chest irradiation are more prone to suffer from cyclophosphamide-induced cardiotoxicity [3]. Liver damage was also reported [73, 74].

Cyclophosphamide compromises calcium accumulation by heart or liver mitochondria, which can almost be restored by CyA [75]. As the increases in the levels of serum AST, serum ALT, glucose-6-phosphate dehydrogenase and creatine phosphokinase induced by cyclophosphamide can also be attenuated by the simultaneous administration of CyA, the induction of MPT is closely related to the hepatotoxicity and cardiotoxicity promoted by cyclophosphamide (Fig.1) [75].

Ifosfamide, another nitrogen mustard, is one of the most commonly implicated drugs in kidney injury [76]. Using mitochondria isolated from the kidney of rats treated with ifosfamide, it was shown that this alkylating agent significantly inhibits complex I, resulting in NADH elevation and NAD+ depletion, and Krebs cycle impairment (Fig.1) [77]. Among the ifosfamide metabo‐ lites, only chloroacetaldehyde, which reaches high concentrations in the renal cortex, inhibits complex I, suggesting that this metabolite is responsible for the ifosfamide-induced nephro‐ toxicity [77].

#### *4.3.3. Alkyl sulfonates*

Busulfan is an alkylating drug, which forms DNA intrastrand crosslinks, used in the clinical management of chronic myelogenous leukemia. Although in standard doses busulfan rarely causes liver dysfunction, some cases of hepatotoxicity during busulfan treatment were reported [78-80].

The toxicity of busulfan is thought to involve oxidative stress mechanisms as it promotes decreases in GSH in hepatocytes both *in vivo* and *in vitro* [81]. Considering that glutathione Stransferase inhibitors and antioxidants prevent busulfan toxicity *in vitro*, it is likely that busulfan toxicity requires glutathione conjugation [81] Moreover, the effects of busulfan are strongly exacerbated on GSH-depleted hepatoctyes [81], which may provide a basis to explain the enhanced sensitivity to the liver damaging effects of busulfan under certain circumstances.

#### **4.4. Enzyme inhibitors**

#### *4.4.1. Anthracyclines*

Cisplatin is a rare cause of hepatotoxicity (steatosis and cholestasis) at standard doses, but high doses may lead to liver damage, revealed by abnormal liver tests, especially aspartate amino‐ transferase (AST) and alanine aminotransferase (ALT) [69]. Light microscopic observations confirmed that high doses of cisplatin cause massive hepatotoxicity; alterations at the ultra‐ structural level, including atrophied mitochondria, were also found [70]. Cisplatin induces MPT in rat liver mitochondria [71]. Moreover, cisplatin stimulates state 4 respiration, but does not affect FCCP-uncoupled respiration, suggesting that cisplatin affects mitochondrial bioenergetics by increasing the IMM permeability to protons and not by interfering with respiratory chain complexes activity [71]. Both the induction of MPT and the effects on liver mitochondrial bioenergetics are prevented by thiol group protecting agents, suggesting that changes on the redox-status of thiol groups affect membrane permeability to cations and underlie liver mitochondrial dysfunction [71]. Accordingly, it was proposed that the mecha‐ nism of cisplatin-induced hepatotoxicity involves membrane rigidification, lipid peroxidation, oxidative damage of cardiolipin and protein sulfhydryl groups, as well as decreased GSH/

Cyclophosphamide is an alkylating agent used in the treatment of lymphomas, multiple myeloma, and certain types of leukemia, retinoblastoma, neuroblastoma, ovarian cancer, and breast cancer. Generally, cyclophosphamide does not cause relevant cardiotoxicity, but when it occurs, it appears to be related to a single dose, unlike the anthracyclines [3]. Patients who were previously medicated with anthracyclines or that underwent chest irradiation are more prone to suffer from cyclophosphamide-induced cardiotoxicity [3]. Liver damage was also

Cyclophosphamide compromises calcium accumulation by heart or liver mitochondria, which can almost be restored by CyA [75]. As the increases in the levels of serum AST, serum ALT, glucose-6-phosphate dehydrogenase and creatine phosphokinase induced by cyclophosphamide can also be attenuated by the simultaneous administration of CyA, the induction of MPT is closely related to the hepatotoxicity and cardiotoxicity promoted by

Ifosfamide, another nitrogen mustard, is one of the most commonly implicated drugs in kidney injury [76]. Using mitochondria isolated from the kidney of rats treated with ifosfamide, it was shown that this alkylating agent significantly inhibits complex I, resulting in NADH elevation

lites, only chloroacetaldehyde, which reaches high concentrations in the renal cortex, inhibits complex I, suggesting that this metabolite is responsible for the ifosfamide-induced nephro‐

Busulfan is an alkylating drug, which forms DNA intrastrand crosslinks, used in the clinical management of chronic myelogenous leukemia. Although in standard doses busulfan rarely

depletion, and Krebs cycle impairment (Fig.1) [77]. Among the ifosfamide metabo‐

GSSG ratio, ATP, GSH and NADPH [72].

32 Toxicology Studies - Cells, Drugs and Environment

*4.3.2. Nitrogen mustards*

reported [73, 74].

and NAD+

toxicity [77].

*4.3.3. Alkyl sulfonates*

cyclophosphamide (Fig.1) [75].

Doxorubicin is used in the clinical management of a wide range of cancers, and particularly in breast cancer treatment. The anticancer activity of doxorubicin involves its intercalation into DNA and disruption of topoisomerase-II-mediated DNA repair, as well as the generation of ROS that lead to lipid peroxidation, as well as membrane and DNA damage [82]. However, its therapeutic use is limited by its side-effects, mainly the dose-dependent myelosuppression and the cumulative and irreversible cardiotoxicity, which in the most severe forms may lead to patient death [83].

Acute cardiotoxicity occurs in less than 1 % of the patients immediately after infusion and is usually reversible; the early-onset chronic progressive form affects 1.6–2.1 % of patients, during therapy or within the first year after treatment; the late-onset chronic progressive form occurs after one year of completion of therapy in 1.6–5 % of patients, supporting the need of longterm follow-up [3]. Doxorubicin causes myocardial damage as shown by the increase in serum levels of AST, ALT, LDH isoenzyme and creatine phosphokinase isoenzyme [84]. Doxorubicininduced cardiotoxicity etiology seems complex, and several effects may be involved, triggering a domino effect [83]. Among the several mechanisms postulated, the induction of oxidative stress is the most widely accepted. The univalent reduction of the tetracyclic ring of anthra‐ cycline by complex I generates a semiquinone free radical; the unpaired electron of this semiquinone is transferred to oxygen, forming the superoxide radical, while the tetracyclic ring returns to the parent quinone [85]. The free radicals generated are thought to be related to the interference with calcium homeostasis and bioenergetic functions, lipid peroxidation, and mtDNA damage, which play a key role in the pathogenesis of doxorubicin cardiotoxicity (Fig.1).

Heart mitochondria isolated from rats treated with doxorubicin present decreased state 3 respiration and respiratory control ratio (RCR), whereas the state 4 respiration is not affected [86-88]; complex I activity is also inhibited [87]. Besides affecting nuclear DNA, doxorubicin damages mtDNA [89-91] and decreases its content in human hearts [90]. Accordingly, doxorubicin-exposed human hearts show low activity of complex I and IV (encoded by mtDNA) but not of complex II (exclusively encoded by nuclear DNA) [90]. The higher levels of superoxide in doxorubicin-exposed hearts correlate negatively with mtDNA content and with the activities of respiratory chain complexes encoded by mtDNA [90]. The damage leading to mtDNA adducts, as well as the higher rate of ROS formation and depression of GSH in heart tissue, persist for several weeks after cessation of doxorubicin treatment [92, 93].

Furthermore, mitochondria isolated from the heart of rats treated with doxorubicin present diminished ability to accumulate calcium [84, 86, 87, 94]. Similar effects were reported in mitochondria isolated from doxorubicin-treated human atrial trabeculae, and were shown to be reversed by CyA [95]. Considering that the decrease in left ventricular fractional survival promoted by doxorubicin is improved by the simultaneous administration of CyA, it seems that doxorubicin-induced heart damage is closely related to the induction of MPT pore opening [84, 96]. As discussed in section 3.3, oxidative stress is a major factor regulating MPT and doxorubicin leads to the oxidation of mitochondrial glutathione and to the accumulation of membrane disulfides, which may contribute to MPT induction. On the other hand, it has been proposed that the ANT is a key target for doxorubicin, as following doxorubicin treatment the amount of ANT protein and its active content are reduced in rats [94, 97]. The effects of doxorubicin on the ANT explain both the MPT induction and the effects on mitochondrial respiration [97]. Indeed, the decrease in state 3 respiration observed in heart mitochondria isolated from doxorubicin-treated rats is partially reversed by CyA or dithiothreitol, but not by trolox, suggesting that the toxic effects of doxorubicin on mitochondrial bioenergetics are at least in part a consequence of MPT induction and involve changes in the redox state of thiol groups [88]. Noteworthy, among several agents, including antioxidants, CyA was the only agent that was able to reverse the doxorubicin-induced alterations in the calcium accumulation capacity when added *ex vivo* [94]. Although the triggering of MPT by doxorubicin may initially involve the oxidation of regulatory components of the MPT pore megacomplex, once it occurs thiol protecting agents are unable to restore the pore to its original closed state [94, 98]. Therefore, antioxidants may be useful in the preventive setting, as discussed in section 5. Moreover, by increasing the generation of free radicals, doxorubicin also significantly enhan‐ ces lipid peroxidation, as well as alterations in proteins and biomolecules that act as signaling molecules [99-101].

Altogether, the results obtained so far suggest that the persistent nature of doxorubicin cardiotoxicity reflects a self-perpetuating mechanism, where mtDNA alterations accumulate, leading to a damaged respiratory chain and decreased calcium loading ability; the defective respiratory chain further enhances ROS generation and mtDNA insult (Fig.1) [83, 98].

The heart is the main target of doxorubicin toxicity, due to the abundance of mitochondria in heart tissue, the elevated rate of oxygen consumption and the lower antioxidant defenses [83]. However, mitochondrial dysfunction has also been observed in other tissues. Indeed, in isolated mitochondrial fractions from the brain of rats treated with doxorubicin, the thiobar‐ bituric acid-reactive substances and the vitamin E levels are increased, whereas the reduced glutathione content is diminished [102]. In addition, doxorubicin increases the sensibility of brain mitochondria to MTP pore opening [102]. The use of doxorubicin was also associated with a higher risk for developing hepatotoxicity among breast cancer patients [103]. Liver mitochondria isolated from doxorubicin-treated rats present decreased RCR and the activity of complex IV is inhibited [107]. In addition, light microscopic observations confirmed that doxorubicin at high doses caused massive liver injury; alterations at the ultrastructural level, such as atrophied mitochondria, were shown [70].

#### *4.4.2. Topoisomerase inhibitors*

leading to mtDNA adducts, as well as the higher rate of ROS formation and depression of GSH in heart tissue, persist for several weeks after cessation of doxorubicin treatment [92, 93].

Furthermore, mitochondria isolated from the heart of rats treated with doxorubicin present diminished ability to accumulate calcium [84, 86, 87, 94]. Similar effects were reported in mitochondria isolated from doxorubicin-treated human atrial trabeculae, and were shown to be reversed by CyA [95]. Considering that the decrease in left ventricular fractional survival promoted by doxorubicin is improved by the simultaneous administration of CyA, it seems that doxorubicin-induced heart damage is closely related to the induction of MPT pore opening [84, 96]. As discussed in section 3.3, oxidative stress is a major factor regulating MPT and doxorubicin leads to the oxidation of mitochondrial glutathione and to the accumulation of membrane disulfides, which may contribute to MPT induction. On the other hand, it has been proposed that the ANT is a key target for doxorubicin, as following doxorubicin treatment the amount of ANT protein and its active content are reduced in rats [94, 97]. The effects of doxorubicin on the ANT explain both the MPT induction and the effects on mitochondrial respiration [97]. Indeed, the decrease in state 3 respiration observed in heart mitochondria isolated from doxorubicin-treated rats is partially reversed by CyA or dithiothreitol, but not by trolox, suggesting that the toxic effects of doxorubicin on mitochondrial bioenergetics are at least in part a consequence of MPT induction and involve changes in the redox state of thiol groups [88]. Noteworthy, among several agents, including antioxidants, CyA was the only agent that was able to reverse the doxorubicin-induced alterations in the calcium accumulation capacity when added *ex vivo* [94]. Although the triggering of MPT by doxorubicin may initially involve the oxidation of regulatory components of the MPT pore megacomplex, once it occurs thiol protecting agents are unable to restore the pore to its original closed state [94, 98]. Therefore, antioxidants may be useful in the preventive setting, as discussed in section 5. Moreover, by increasing the generation of free radicals, doxorubicin also significantly enhan‐ ces lipid peroxidation, as well as alterations in proteins and biomolecules that act as signaling

Altogether, the results obtained so far suggest that the persistent nature of doxorubicin cardiotoxicity reflects a self-perpetuating mechanism, where mtDNA alterations accumulate, leading to a damaged respiratory chain and decreased calcium loading ability; the defective

The heart is the main target of doxorubicin toxicity, due to the abundance of mitochondria in heart tissue, the elevated rate of oxygen consumption and the lower antioxidant defenses [83]. However, mitochondrial dysfunction has also been observed in other tissues. Indeed, in isolated mitochondrial fractions from the brain of rats treated with doxorubicin, the thiobar‐ bituric acid-reactive substances and the vitamin E levels are increased, whereas the reduced glutathione content is diminished [102]. In addition, doxorubicin increases the sensibility of brain mitochondria to MTP pore opening [102]. The use of doxorubicin was also associated with a higher risk for developing hepatotoxicity among breast cancer patients [103]. Liver mitochondria isolated from doxorubicin-treated rats present decreased RCR and the activity of complex IV is inhibited [107]. In addition, light microscopic observations confirmed that

respiratory chain further enhances ROS generation and mtDNA insult (Fig.1) [83, 98].

molecules [99-101].

34 Toxicology Studies - Cells, Drugs and Environment

Etoposide is a podophyllotoxin derivative used in the treatment of several cancers that acts as a topoisomerase II inhibitor. Cases of hepatic injury were reported using either standard doses [104] or high-dose regimens [105]. Etoposide induces calcium-dependent MPT in rat liver mitochondria [106]. Since the etoposide-induced MPT is prevented by several antioxidants, it was proposed that etoposide triggers MPT pore opening through the generation of oxidant species, which is further corroborated by the ability of antioxidants to prevent the apoptosis promoted by etoposide (Fig.1) [107]. Therefore, the generation of oxidant species that are able to induce mitochondrial dysfunction may contribute to hepatic injury.

#### **4.5. Antimicrotubules — Taxanes**

Paclitaxel is a taxane-derived drug used in monotherapy or in combination with other agents, for the treatment of ovarian, breast and advanced non-small cell lung cancers, and AIDSrelated Kaposi's sarcoma.

Painful peripheral neuropathy is the major dose-limiting side-effect of paclitaxel therapy, with the major drawback that the pain and sensory abnormalities can persist for months or years. Moreover, it may turn the patients unable to complete optimal chemotherapy schedules thus potentially compromising the treatment efficacy [108].

The involvement of mitochondrial dysfunction in paclitaxel-induced pain is suggested by the presence of swollen and vacuolated neuronal mitochondria [109]. Earlier investigations demonstrated that paclitaxel promotes a CyA-sensitive swelling in liver, kidney, heart, and brain mitochondria; the highest degree and slope of the swelling are observed in liver mitochondria, whereas the lowest are detected in brain mitochondria [110].

Using a rat model of paclitaxel-induced pain, it was shown that the non-specific ROS scavenger N-tert-butyl-α-phenylnitrone significantly decreases paclitaxel-induced mechanical hyper‐ sensitivity, whereas the superoxide selective scavenger 4-hydroxy-2, 2, 6, 6-tetramethylpiper‐ idine-1-oxyl (TEMPOL) does not present significant effects [108]. Therefore, the authors suggest that ROS are involved in the development and maintenance of paclitaxel-induced pain, although such effects cannot be attributed to superoxide radicals alone [108]. Moreover, rat sciatic nerve samples taken after induction of painful peripheral neuropathy with paclitaxel exhibit significant impairment of both complex I- and complex II-mediated respiration and deficits in ATP synthesis; the mitochondrial dysfunction promoted by paclitaxel is abrogated by acetyl-l-carnitine, again supporting that paclitaxel promotes oxidative damage [65].

Paclitaxel is extensively excreted by the liver, and therefore its administration to patients with liver impairment should be handled with care [69]. Alterations of liver functions are seen in 4-17 % of patients treated with doses up to 190 mg/m2 , but they can occur in 16-37 % of patients taking higher doses [69].

In isolated liver mitochondria, paclitaxel induces large amplitude swelling, the dissipation of mitochondrial membrane potential and the release of cytochrome c; these effects are inhibited by CyA, suggesting that paclitaxel induces MPT pore opening [110]. Paclitaxel also signifi‐ cantly increases complex IV-mediated ROS production (Fig.1) [110]. Paclitaxel does not inhibit mitochondrial respiration, but ROS formation is abolished by complex IV inhibitors, suggest‐ ing that paclitaxel promotes ROS production not by inhibiting the respiratory complexes, but through an effect on complex IV [110]. The abrogation of ROS formation does not prevent paclitaxel-induced MPT, suggesting that the induction of MPT is not secondary to enhanced ROS generation [110]. The combination with doxorubicin enhances the induction of oxidative stress [111].

The cardiotoxicity promoted by paclitaxel is usually represented by subclinical sinus brady‐ cardia (approximately 30 % of patients), but more severe conditions were also reported [3]. A recent study suggests that microtubule disorganization in cardiac myocytes promoted by paclitaxel leads to MTP pore opening [112]. However, when isolated mitochondria are exposed to paclitaxel, no significant effects are detected; the authors suggested that paclitaxel does not promote MPT due to a direct effect on mitochondria [112]. However, it must be noted that lower concentrations were used in the latter study in comparison with previous work using liver mitochondria [110, 112].

Therefore, both the induction of MPT and mitochondrial ROS production can contribute to paclitaxel side effects in the nerve, liver, kidney and heart.

#### **4.6. Antimetabolites**

#### *4.6.1. Folate antagonists*

Methotrexate is a folic acid antagonist widely used in the treatment of leukemia and other malignancies. Gastrointestinal toxicity and liver function abnormalities are common in patients taking methotrexate and the use of methotrexate in patients with history of liver disease is not advisable [113].

In liver mitochondria, methotrexate promotes a significant rise in superoxide radical formation, as well as in lipid peroxidation, whereas the GSH levels are decreased [114]. Likewise, methotrexate significantly impairs the function of isolated heart mitochondria by promoting lipid peroxidation, mitochondrial swelling and by inhibiting complex I, II and IV activities [115].

Methotrexate administration also leads to small intestinal injury and damages in enterocyte mitochondria, as shown by the decrease in the RCR, an indicator of mitochondrial function [116]. Moreover, the activities of complexes II and IV are markedly decreased in enterocyte mitochondria, suggesting that the deleterious effects promoted by methotrexate on enterocyte mitochondria can compromise ATP synthesis (Fig.1) [116], thereby leading to the gastrointes‐ tinal toxicity seen in patients.

#### *4.6.2. Purine analogs*

In isolated liver mitochondria, paclitaxel induces large amplitude swelling, the dissipation of mitochondrial membrane potential and the release of cytochrome c; these effects are inhibited by CyA, suggesting that paclitaxel induces MPT pore opening [110]. Paclitaxel also signifi‐ cantly increases complex IV-mediated ROS production (Fig.1) [110]. Paclitaxel does not inhibit mitochondrial respiration, but ROS formation is abolished by complex IV inhibitors, suggest‐ ing that paclitaxel promotes ROS production not by inhibiting the respiratory complexes, but through an effect on complex IV [110]. The abrogation of ROS formation does not prevent paclitaxel-induced MPT, suggesting that the induction of MPT is not secondary to enhanced ROS generation [110]. The combination with doxorubicin enhances the induction of oxidative

The cardiotoxicity promoted by paclitaxel is usually represented by subclinical sinus brady‐ cardia (approximately 30 % of patients), but more severe conditions were also reported [3]. A recent study suggests that microtubule disorganization in cardiac myocytes promoted by paclitaxel leads to MTP pore opening [112]. However, when isolated mitochondria are exposed to paclitaxel, no significant effects are detected; the authors suggested that paclitaxel does not promote MPT due to a direct effect on mitochondria [112]. However, it must be noted that lower concentrations were used in the latter study in comparison with previous work using

Therefore, both the induction of MPT and mitochondrial ROS production can contribute to

Methotrexate is a folic acid antagonist widely used in the treatment of leukemia and other malignancies. Gastrointestinal toxicity and liver function abnormalities are common in patients taking methotrexate and the use of methotrexate in patients with history of liver

In liver mitochondria, methotrexate promotes a significant rise in superoxide radical formation, as well as in lipid peroxidation, whereas the GSH levels are decreased [114]. Likewise, methotrexate significantly impairs the function of isolated heart mitochondria by promoting lipid peroxidation, mitochondrial swelling and by inhibiting complex I, II and

Methotrexate administration also leads to small intestinal injury and damages in enterocyte mitochondria, as shown by the decrease in the RCR, an indicator of mitochondrial function [116]. Moreover, the activities of complexes II and IV are markedly decreased in enterocyte mitochondria, suggesting that the deleterious effects promoted by methotrexate on enterocyte mitochondria can compromise ATP synthesis (Fig.1) [116], thereby leading to the gastrointes‐

stress [111].

liver mitochondria [110, 112].

36 Toxicology Studies - Cells, Drugs and Environment

**4.6. Antimetabolites**

*4.6.1. Folate antagonists*

IV activities [115].

disease is not advisable [113].

tinal toxicity seen in patients.

paclitaxel side effects in the nerve, liver, kidney and heart.

6-Mercaptopurine is an orally administered immunosuppressive drug used to treat acute lymphocytic leukemia. 6-Mercaptopurine is converted to its active metabolites, the 6-thiogui‐ nine nucleotides, or inactivated by xanthine oxidase or by thiopurine methyltransferase to 6 thiouric acid or 6-methylmercaptopurine, respectively [117]. Liver injury is an important adverse effect of 6-mercaptopurine, with an estimated frequency of liver test abnormalities within 1-9 % range [118]. Clinically relevant concentrations of 6-mercaptopurine are toxic to rat hepatocyte cultures by a mechanism that involves oxidative stress and ATP depletion (Fig. 1) [119]. The decreased rat hepatocytes viability promoted by 6-mercaptopurine is nearly prevented by allopurinol (xanthine oxidase inhibitor) together with trolox (vitamin E analog), implying xanthine oxidase-mediated metabolism of the thiopurines and oxidative stress in the hepatotoxicity promoted by 6-mercaptopurine [119].
