**6. Concluding remarks**

pharmacological strategies aiming to avoid alterations that may prove deleterious both in the short and in the long term. Considering that some of these effects are irreversible or cumulative,

As discussed in the previous section, oxidative stress has been established as one of the primary cause of mitochondrial dysfunction and toxicity induced by anticancer agents and, therefore, several antioxidants have been tested *in vitro* and *in vivo* as a prophylactic measure. In particular, naturally occurring antioxidants have been investigated as therapeutic adjuvants, as they are considered safe and well-tolerated, and may afford protection against cancer

Alpha-lipoic acid affords protection against the neurotoxic effects promoted by cisplatin and paclitaxel through its antioxidant and mitochondrial regulatory functions [135]. The toxic effects promoted by cisplatin on rat liver mitochondria are also prevented by thiol group protecting agents [71]. Curcumin, which has anti-inflammatory and anticancerous properties, counteracts the mitochondrial lipid peroxidation and GSH levels alterations in mitochondria isolated from the brain and liver of rats treated with cisplatin, suggesting that it can abrogate the toxic effects of cisplatin on brain and liver [136]. Likewise, epicatechin prevents the renal damage and mitochondrial dysfunction promoted by cisplatin by decreasing oxidative stress; noteworthy, epicatechin does not compromise the antitumor

The etoposide-induced MPT is prevented by ascorbate, the primary reductant of the phenoxyl

An *in vitro* study demonstrated that Vitamin E decreases the oxidative stress induced by methotrexate in rat heart mitochondria and thereby minimizes mitochondrial dysfunction [115]. Likewise, the administration of lipoic acid decreases oxidative stress induced by

Acetyl-L-carnitine completely blocks the effects of bortezomib on mitochondria and pain [134]. Strategies to prevent doxorubicin-induced cardiotoxicity are probably the best studied, given the significant number of patients affected and the impact on the overall success of the treatment. Many studies reported that antioxidants could afford cardioprotection against doxorubicin therapy. The broad antioxidant resveratrol markedly ameliorates the cardiac dysfunction promoted by doxorubicin, while the ROS generation is decreased, and gluta‐ thione, superoxide dismutase and catalase activities are improved [137]. Also, flavonoids, and particularly 7-monohydroxyethylrutoside, protect against the cardiac toxic effects promoted by doxorubicin both *in vitro* and *in vivo* [138]. In addition, 7-monohydroxyethylrutoside does not compromise the antitumor activity of doxorubicin in human ovarian cell lines and in the corresponding mouse xenograft models, and even inhibits the overexpression of adhesion molecules promoted by doxorubicin on vascular endothelial cells [138]. The combination of doxorubicin and vitamin E-succinate cooperates to induce apoptosis in human gastric cancer cells, by promoting doxorubicin influx and suppressing its efflux [139]. On the other hand, vitamin E also aggravates the heart damage promoted by doxorubicin in P388 tumor-bearing

it is desirable to prevent these events when planning the therapy of cancer patients.

treatment-related toxicity by improving mitochondrial functions.

radicals generated by etoposide, and by thiol protecting agents [107].

methotrexate, which affects liver mitochondrial function [114].

actions of cisplatin in HeLa cells [67].

40 Toxicology Studies - Cells, Drugs and Environment

mice [140].

Considering the key role played by mitochondria in cell survival and death, the pharmaco‐ logical modulation of mitochondrial activity has been investigated in cancer therapy [13, 158]. It is thought that this strategy may overcome the resistance mechanisms related with conven‐ tional chemotherapy that do not target mitochondria directly, but interfere with signaling pathways which lie upstream of mitochondria and that are frequently deregulated in cancer [158]. However, the targeting of mitochondria as a therapeutic strategy is often compromised by the absence of significant pathophysiological differences between mitochondria in normal and malignant cells, leading to reduced selectivity of drugs targeting mitochondria. Therefore, the actions that are beneficial in cancer cells may, in contrast, underlie some of the severe toxic effects promoted by these agents.

Indeed, the induction of mitochondrial damage is an important contributor for some of the most well-known toxic effects of anticancer agents, namely the liver injury promoted by tamoxifen [159], the cardiotoxicity of doxorubicin or the cisplatin-induced neuropathy and nephrotoxicity. Organ dysfunction has a significant impact on the treatment outcomes and, therefore, the better understanding of the mechanisms of toxicity may unveil strategies to limit, or preferably to prevent, the incidence of these events and thereby improve the overall clinical success.

The recognition that mitochondrial dysfunction plays a key role in drug-induced toxicity may contribute to identify the drugs that are more likely to lead to such effects at an early stage. In this context, the use of isolated mitochondria fractions is a valuable tool to predict drug safety, since it provides relevant information while allowing to reduce the number of laboratory animals and the costs of preclinical studies [8].

On the other hand, our current knowledge does not allow to predict the idiosyncratic injury related with drug-induced mitochondrial dysfunction. It seems that genetic, metabolic and environmental factors that impair mitochondrial function can add their effects to those of anticancer drugs, compromising mitochondrial function to an extent where manifestations start to occur [17]. Therefore, therapeutic drug monitoring is mandatory. Furthermore, as organ damage may become apparent months or even years after the completion of the treatment (e.g. late-onset doxorubicin toxicity) the need of long-term follow-up is reinforced.

Finally, future studies should aim to develop strategies which are able to afford protection against both the short-term and long-term effects of anticancer drugs and without compro‐ mising their antitumor activity. Although antioxidants showed promise in *in vitro* studies, inconsistent results and failure in clinical trials turn the use of antioxidants as adjuvants in cancer therapy hardly consensual [7, 83]. However, in this context, we need to take into consideration that antioxidants may present different intracellular localization patterns and interfere with normal redox signaling pathways in specific cell compartments; an approach involving the targeted delivery of antioxidants to mitochondria can possibly provide better outcomes [7, 83]. Moreover, there are important differences between *in vitro* and *in vivo* toxicities and between animal models and humans. The different drug metabolism and clearance, as well as the asymmetries in redox regulation may account for the difficulty in translating these strategies into human subjects [83].

In conclusion, studies in suitable animal models are vital for a better understanding of the mechanisms underlying drug toxicity and the benefits of strategies aiming to prevent mito‐ chondrial damage. So far most studies have used animal models devoid of tumors, which add an extra physiological burden that may influence the effects of drugs [83]. Moreover, as described in the previous section, some of the toxic effects on mitochondria are observed in several organs, including the liver and kidneys, which may compromise both the pharmaco‐ kinetics and the efficacy of the anticancer drugs, but also the benefit of therapeutic adjuvants aiming to protect the mitochondria. These observations emphasize the importance of per‐ forming *in vivo* studies in relevant models, as well as the crucial importance of the clinical control and therapeutic drug monitoring of patients treated with anticancer drugs.
