Biomarkers of Mitochondrial Dysfunction and Toxicity

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55 Biomarkers of Mitochondrial Dysfunction and Toxicity Carlos M. Palmeira, Joa˜o S. Teodoro, Rui Silva, Anabela P. Rolo Center for Neurosciences and Cell Biology of the University of Coimbra and Department of Life Sciences of the University of Coimbra Largo Marqueˆs de Pombal, Coimbra, Portugal

INTRODUCTION Mitochondria play a central role in the life and death of cells. They are not only the mere center for energy metabolism and ATP generation but are also the prime location for different catabolic and anabolic processes, calcium fluxes, and various signaling pathways, while also playing major role in cell life-defining processes such as apoptosis. Mitochondria maintain cellular homeostasis by interacting with reactive oxygenenitrogen species and responding adequately to different stimuli. In this context, the interaction of pharmacological agents with mitochondria is an aspect of molecular biology that is too often disregarded, not only in terms of toxicology but also from a pharmaceutical point of view, especially when considering the potential therapeutic applications related to the modulation of mitochondrial activity. Numerous works have shown that mitochondria are a major toxicological target, with their dysfunction being a major mechanism of drug-induced injury. The aim of this chapter is to highlight the role of mitochondria and the modulation of mitochondrial activities in pharmacology and toxicology and also to stress some of the potential therapeutic approaches. In recent years, there has been extraordinary progress in mitochondrial science that has further outlined the critical role of these organelles in cell biology, pathophysiology, and the diagnosis and therapeutic treatment of different human diseases, such as ischemic diseases, diabetes, some forms of neurodegeneration, and cancer (Duchen, 2004b; Scatena et al., 2007; Giorgi et al., 2012). Mitochondrial physiology and pathophysiology is notably complex, and the role of mitochondria in bioenergetics is also linked, as mentioned earlier, to other essential functions, such as anabolic metabolism,

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00055-4

the balance of redox potential, cell death and differentiation, and mitosis. In addition to these basic functions, mitochondria are associated with more specialized cell activities, including calcium homeostasis and thermogenesis, reactive oxygen species (ROS) and reactive nitrogen species signaling, maintenance of ion channels, and the transport of metabolites. Consequently, the basis of different congenital mitochondrial diseases on a molecular level is equally complex and heterogeneous, making mitochondrial pathophysiology difficult to investigate (Hamm-Alvarez and Cadenas, 2009; Cardoso et al., 2010). This field is made even more challenging by recent evidence that suggests mitochondrial structure and function is dynamic. Specifically, mitochondria possess many interesting properties, such as the ability to fuse or divide, move along microtubules and microfilaments, or undergo turnover (Westermann, 2010; Michel et al., 2012), and these unique properties are often overlooked in research. Undoubtedly, much is still unknown about the mutual interactions between mitochondrial energetics, biogenesis, dynamics, and degradation (Detmer and Chan, 2007), and the contribution of these interactions to mitochondrial toxicology and pharmacology.

MITOCHONDRIAL FUNCTION: GENERAL OVERVIEW The mitochondrion consists of four main structures or compartments: two membranes, the intermembrane space, and the matrix within the inner membrane. The mitochondrial outer membrane (MOM) separates the cytosol from the intermembrane space. The MOM is responsible for interfacing with the cytosol and its interactions with cytoskeletal elements, which are important for

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the movement of mitochondria within a cell. This mobility is essential for the distribution of mitochondria during cell division and differentiation. The mitochondrial inner membrane (MIM) separates the intermembrane space from the matrix. The foldings of the MIM toward the matrix (cristae) serve to increase the surface area of this membrane. Mitochondria also move along intermediate actin filaments, using kinesin and dynein proteins. The MIM hosts the most important redox reactions converting the energy of nutrients into ATP. These reactions are catalyzed by the mitochondrial electron transport chain (ETC), which transports electrons from several substrates to oxygen, in the complex multistep process termed as mitochondrial respiration. According to the chemiosmotic theory, mitochondrial respiration generates a transmembrane potential (DJm) across the inner membrane, which is used by ATP synthase to phosphorylate ADP. The MIM is normally virtually impermeable to protons and other ions, and this solute barrier function of the MIM is critical for energy transduction. Permeabilization of the MIM dissipates the DJm and thereby uncouples the process of respiration from the ATP synthase, halting mitochondrial ATP production (Kushnareva and Newmeyer, 2010). Hence, the free energy of respiration is used to pump protons from the matrix to the intermembrane space (IMS), establishing an electrochemical gradient. Because the MIM displays an extremely low passive permeability to protons, an electrochemical gradient (DmHþ) is built across the membrane. The electrochemical gradient is the sum of two components: the proton concentration difference and the electrical potential difference across the membrane. The estimated magnitude of the proton electrochemical gradient is about 220 mV (negative inside), and under physiological conditions most of the gradient is in the form of electrical potential difference. The proton gradient is converted in ATP by the F1F0-ATP synthase. F1F0-ATP synthase couples the transport of the protons back to the matrix with the phosphorylation of ADP to ATP. Inefficient electron transfer through complexes IeIV causes human diseases in part not only because of loss of energy generation capacity but also of insults to the various enzymes (particularly complexes I, II, and III) induce production of toxic ROS. Defects of complex Vare also a cause of mitochondrial dysfunction (Schapira, 2006; Wu et al., 2010a; Abramov et al., 2011). It has also been reported that the deterioration of mitochondrial function underlies common metabolic-related diseases (Rolo and Palmeira, 2006; Palmeira et al., 2007; Turner and Heilbronn, 2008), and several studies have identified compromised oxidative metabolism, altered mitochondrial structure and dynamics, and impaired biogenesis and gene expression in insulin resistance or type 2 diabetes (T2DM) models (Cheng et al., 2010; Rolo et al., 2011; Gomes et al., 2012; Dela and Helge, 2013; Teodoro et al., 2013). In addition to the process of ATP formation, mitochondria are highly dynamic organelles that have been

implicated in the regulation of a great and increasing number of physiological processes. Cells need energy not only to support their vital functions but also to die gracefully, through programmed cell death, or apoptosis (Kushnareva and Newmeyer, 2010). Execution of an apoptotic program includes energy-dependent steps, including kinase signaling, formation of the apoptosome, and effector caspase activation. Furthermore, mitochondrial regulation is also present beyond cell death mechanisms. Indeed, besides oxidative ATP production, mitochondria assume other functions such as heme synthesis, b-oxidation of free fatty acids, metabolism of certain amino acids, production of free radical species, formation and export of Fe/S clusters, and iron metabolism and play a crucial role in calcium homeostasis (Duchen, 2004a; Michel et al., 2012). In addition, initially described as a key checkpoint of intrinsic programmed cell death, accumulating data point to mitochondria as a central platform involved in many cellular pathways, such as those recently highlighted participating in the innate immune response (West et al., 2011) or its lipidic contribution to autophagosomal membrane genesis during starvation-induced autophagy (Hailey et al., 2010). Still, the regulatory roles of mitochondria over normal physiology include the transduction pathway that underlies the secretion of insulin in response to glucose by pancreatic b-cells and in the evaluation of oxygen tension necessary for sensing oxygen in the carotid body and the pulmonary vasculature. Mitochondria also house key enzyme systems quite distinct from those required for intermediary metabolismdthe rate-limiting enzymes in steroid biosynthesis and even the carbonic anhydrase required for acid secretion in the stomach (Duchen, 2004b). By accumulating calcium when cytosolic calcium levels are high, mitochondria play subtle roles in coordinating the complexities of intracellular calcium-signaling pathways, at least in some cell types, in which their contribution may be extremely important in the finer aspects of cell regulation. The physiological “uncoupling” of mitochondria plays a central role as a heat-generating mechanism in nonshivering thermogenesis in young and small mammals. It has also been suggested that the production of free radical species by mitochondria might play a key role as a signaling mechanismdfor example, in the regulation of ion-channel activities and also in initiating cytoprotective mechanisms in stressed cells (Michel et al., 2012).

MITOCHONDRIAL TOXICITY Mitochondrial dysfunction is a fundamental mechanism in the pathogenesis of several significant toxic effects in mammals, especially those associated with the liver, skeletal and cardiac muscle, and the central nervous system. These changes can also occur as part

XENOBIOTICS AND MITOCHONDRIAL DYSFUNCTION

of the natural aging process and have been linked to cellular mechanisms in several human disease states including Parkinson’s and Alzheimer’s diseases, as well as ischemic perfusion injury and the effects of hyperglycemia in diabetes mellitus (Amacher, 2005). Knowledge of the effects of xenobiotics on mitochondrial function has expanded to the point that chemical structure and properties can guide the pharmaceutical scientist in anticipating mitochondrial toxicity. Recognition that maintenance of the mitochondrial membrane potential is essential for normal mitochondrial function has resulted in the development of predictive cell-based or isolated mitochondrial assay systems for detecting these effects with new chemical entities. The homeostatic role of some uncoupling proteins, differences in mitochondrial sensitivity to toxicity, and the pivotal role of mitochondrial permeability transition (MPT) as the determinant of apoptotic cell death are factors that underlie the adverse effects of some drugs in mammalian systems. To preserve mitochondrial integrity in potential target organs during therapeutic regimens, a basic understanding of mitochondrial function and its monitoring in the drug development program are essential. At the mitochondrial level, there are several potential drug targets that can lead to toxicity, but a real clinical counterpart has been demonstrated only for a few of them. Recently, antiviral nucleoside analog have shown mitochondrial toxicity through the inhibition of DNA polymerase gamma. Other drugs targeted to different components of the mitochondrial channels can disrupt ion homeostasis or affect the MPT pore. Many molecules are known as inhibitors of the mitochondrial ETC, interfering with one or more of the complexes in the respiratory chain. Some drugs, including nonsteroidal antiinflammatory drugs (NSAIDs), may lead to uncoupling of oxidative phosphorylation, whereas the mitochondrial toxicity of other drugs seems to depend on the production of free radicals, although this mechanism has yet to be clearly defined. Besides toxicity, other drugs have been targeted toward mitochondria to treat mitochondrial dysfunctions. A clear example is the recent development of drugs that target the mitochondria of cancer cells to trigger apoptosis or necrosis, thus promoting cell death and fighting cancer (Rohlena et al., 2011).

XENOBIOTICS AND MITOCHONDRIAL DYSFUNCTION Mitochondria, because of their central role in metabolism and cell function, have been often used to assess chemical-induced toxicity. Organophosphorus (OPs) pesticides are a class of widely used pesticides in agriculture and in domestic uses. Mitochondria as a site of

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cellular oxygen consumption and energy production can be a target for OP poisoning as a noncholinergic mechanism of OPs toxicity (Gupta et al., 2001a,b; Karami-Mohajeri and Abdollahi, 2013). Some toxic effects of OPs arise from the dysfunction of mitochondrial oxidative phosphorylation through alteration of the activities of all respiratory complexes and disruption of the mitochondrial membrane. Reduction of ATP synthesis or induction of its hydrolysis can impair the cellular metabolism. The OPs perturb cellular and mitochondrial antioxidant defenses, ROS generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome c released from mitochondria and consequent activation of caspases. Mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, and electron donors and increasing the cytosolic ATP level. Moreover, other organophosphates have been reported to induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of the mitochondrial apoptotic pathway, causing neuropathy (Zou et al., 2013). Some compounds used as food additives for growth promotion, such as olaquindox, induce DNA damage and oxidative stress, causing apoptosis in liver cells through the mitochondrial pathway (Zou et al., 2011). More recently, there have been reports of an increase in the frequency of mitochondrial DNA (mtDNA) somatic mutations in lung tissues of fruit growers that had been exposed to pesticides multiple times via inhalation (Wang and Zhao, 2012). The mitochondrial genome is particularly prone to DNA damage, because of its limited DNA repair capabilities, lack of protective histone proteins, and the low tolerance of damaged DNA. Moreover, mitochondria are known to be the major source of reactive oxygen in most mammalian cell types, as well as a major target organelle for oxidative damage (Chomyn and Attardi, 2003). Mitochondrial superoxide and H2O2 can cause direct damage to mitochondrial proteins, resulting in nuclear and mitochondrial genotoxicity (Shen et al., 2005), and initiation of apoptosis. It has been reported that dioxins cause sustained oxidative stress and damage in liver mitochondria from mice exposed to TCDD and in hepatocytes (Senft et al., 2002; Aly and Dome`nech, 2009); thus, mitochondria are also a direct target for dioxin-induced toxicity. In both hepatic mitochondria isolated from TCDDtreated mice and mitochondria incubated in vitro with TCDD, a number of functional alterations have been observed, including a defect in ATP synthesis and increased ROS production (Senft et al., 2002; Shen et al., 2005; Shertzer et al., 2006; Kopf and Walker, 2010). TCDD decreases hepatic ATP levels through changes in mitochondrial F0F1-ATP synthase and

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ubiquinone and generates mitochondrial oxidative DNA damage, which is exacerbated by decreasing mitochondrial reduced (active) glutathione and by inner membrane hyperpolarization. These mitochondrial effects of TCDD are also associated with altered expression of nuclearly encoded mitochondrial genes (Forgacs et al., 2010; Dere et al., 2011), as well as apoptosis induction involving calcium/calmodulin signaling (Kobayashi et al., 2009). In primary hepatocytes, TCDD has been shown to induce an oxidative stress response involving mitochondrial dysfunction (Aly and Dome`nech, 2009) and, in mice, exposure to TCDD causes a loss in mitochondrial membrane potential mediated by AhR-dependent production of ROS (Fisher et al., 2005). Furthermore, previous studies have identified mitochondrial targets of environmental pollutants, namely ROS production and decreased ATP content (Shertzer et al., 2006). Consequently, maintenance of cellular function is strictly dependent on the existence of a healthy population of mitochondria, given that alterations of mitochondrial bioenergetic features by toxicants reduce energetic charge and may ultimately result in cell death. Some detrimental effects of dibenzofuran (DBF), a ubiquitous dioxin-like compound considered to be an environmental pollutant, have already been reported in lung mitochondria (Duarte et al., 2011) and lung cells (Duarte et al., 2012); in addition, some previous studies reported that environmental toxicants induce mitochondrial damage (Palmeira and Madeira, 1997), proving that some pollutants injure mitochondria directly. More recently, siRNA-mediated knockdown of the AhR in lung epithelial cells and fibroblasts was shown to increase sensitivity to smoke-induced apoptosis (Souza et al., 2013), and these effects involved mitochondrial dysfunction, decreased antioxidant enzymes, and oxidative stress.

MITOCHONDRIA AND DISEASE In a clinical setting, research has shown a significant relationship between mitochondrial metabolic abnormalities and tumors found in renal carcinomas, glioblastomas, paragangliomas, or skin leiomyoma, which has led to the discovery of new genes, oncogenes, and oncometabolites involved in the regulation of cellular and mitochondrial energy production with a particular focus on reevaluating the Warburg effect (Fulda et al., 2010; Ralph et al., 2010; Solaini et al., 2011). Furthermore, the examination of rare neurological diseases, such as Charcot-Marie Tooth type 2a, autosomal dominant optic 53 atrophy, lethal mitochondrial and peroxisomal fission, and spastic paraplegia, has suggested the involvement of MFN2, OPA1, DRP1, or paraplegin in

the auxiliary control of mitochondrial energy production (Benard et al., 2010; Du and Yan, 2010; Zhu, 2010). Advances in the understanding of mitochondrial apoptosis have suggested a supplementary role for Bcl-2 or Bax in the regulation of mitochondrial respiration and dynamics, which has led to the investigation of alternative mechanisms of energy regulation (Benard et al., 2010). In addition, different metabolic diseases, such as diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD), and the more general metabolic syndrome underline the role of dysfunctional mitochondria in pathogenesis (Dalgaard, 2011; Rolo et al., 2011).

MITOCHONDRIAL DYSFUNCTION IN DIABETES In a situation of excess nutrients, mitochondrial membrane potential (DJm) can rise to abnormally high levels, with concomitant excessive reduction of the mitochondrial respiratory chain complexes. This occurs because of elevated levels of ATP and low levels of ADP, meaning that the membrane potential generated by the oxidation of substrates is not totally utilized and begins to build up. Although there is a normal buildup of membrane potential, when it reaches high enough values it can lead to extremely dangerous situations. Overreduction means that the electrons obtained from substrate oxidation can no longer reach molecular O2 at complex IV or cytochrome c oxidase to generate H2O. As the vectorial ejection of protons against their gradient is a requirement for electronic transport across the respiratory chain, given a high enough membrane potential, the electronic leap between each complex no longer carries enough energy to transport protons against their enlarged gradient, and for that reason electrons get “stuck” inside the respiratory chain. This is most dangerous, for these proteic complexes are in an altered, unstable conformational state, which they must abandon by getting rid of the electrons to anything that will take them. That turns out to be molecular O2, resulting in the heightened generation of ROS. Given enough time, the abnormal ROS generation overwhelms natural antioxidant defenses and creates mitochondrial damage, further increasing their generation and leading ultimately to cellular and tissue dysfunctions (Fig. 55.1). The increase in nutrients offered without elevated demand for ATP leads to the abnormal augment of membrane potential (DJm). This, in turn, leads to increased ROS generation that, if prolonged enough, causes cellular and mitochondrial damage. By activating PGC-1a (by phosphorylationdAMPK and deacetylationdsirtuins), one can induce the activation of the mitochondrial biogenic program, leading to the generation of more mitochondria. More mitochondria allows for better

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ATP

Sirtuins

AMPK

A c

A c

Cellular and mitochondrial damage

ADP PGC1α

P

PGC1α

P

Transcrip on Factors

Oxida ve damage

ROS

More mitochondria

Reduced ROS genera on

FIGURE 55.1 Role of mitochondria in diabetes and obesity. AMPK, AMP-activated protein kinase; PGC1a, peroxisome proliferator-activated receptor g coactivator 1a; ROS,./ reactive oxygen species; UCP1, uncoupling protein 1.

handling of the excess nutrients, thus reducing ROS generation. Another way to reduce DJm is by mildly uncoupling the oxidative phosphorylation. UCP1 (and other members of the uncoupling protein family) accomplishes this by reducing DJm and generating heat, thus preventing ROS generation. It was found that mitochondria from high-fat fed (HFD) rats were morphologically and structurally altered (Lieber et al., 2004; Kim et al., 2008). These studies demonstrate that there appears to exist a direct correlation between altered mitochondrial functionality and insulin resistance, obesity, and diabetes (Vial et al., 2010). As such, correct mitochondrial structure and function correction could lead to the unveiling of therapeutic strategies to treat obesity and diabetes. The master regulator of mitochondrial biogenesis, the peroxisome proliferatoreactivated receptor g (PPARg) coactivator 1a (PGC1a), is of great necessity for the correct number, structure, and function of mitochondria. The regulation of PGC1a can occur by several means: its expression, phosphorylation, and acetylation status (Fernandez-Marcos and Auwerx, 2011), for example. The ones highlighted are particularly important, for they appear to be dependent on the cell’s energetic status. In fact, it has been shown that sirtuin 1 (SirT1) regulates PGC1a. Sirtuins are a class of NADþ-dependent deacetylases and, as such, their activity on gene

transcription can be classified as a nutrient-sensitive action. Sirtuins’ activity as gene transcription modulators has been explored in various fields of investigation, from aging to obesity, diabetes, and Alzheimer’s, to name a few (Yamamoto et al., 2007). SirT1 effects on metabolic regulation were found on SirT1-null mice, which have decreased insulin release, whereas overexpression of SirT1 has increased insulin response to glucose (Moynihan et al., 2005). Also, SirT1 deacetylates and thus activates PGC1a, correlating directly to improved metabolic status (Nemoto et al., 2005). Curiously, SirT1 decreases uncoupling protein 2 (UCP2) expression, resulting in increased mitochondrial coupling and thus reducing substrate utilization (Moynihan et al., 2005), which makes some sense because elevated NADþ levels activate SirT1 and, as such, the cell has energetic needs and should not waste DJm. Conversely, PPARg’s expression is downregulated by SirT1, resulting in decreased adipogenesis and increased lipolysis (Picard and Auwerx, 2002). SirT1 activation of PGC1a in brown adipocytes leads to increased mitochondrial biogenesis and thus increased thermogenic dissipation of excess nutrients (Lagouge et al., 2006). For further reading on SirT1, readers are encouraged to read the excellent work by Yamamoto et al. (2007). These works are sometimes conflicting and make it difficult to understand the effects of SirT1

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on metabolism. In fact, until very recently, the role of resveratrol (the most famous natural SirT1 activator) was still questioned and not fully understood (Hubbard et al., 2013). SirT1 is the most famous and most studied sirtuin, but it is not the only one. Another extremely important sirtuin is the normally mitochondrial native SirT3. Expression of SirT3, in white and brown adipose tissue (WAT and BAT, respectively), is induced by calorie restriction and cold exposure. One of the most interesting facts about SirT3 is that its constitutive expression causes increased levels of PGC1a and UCP1 expression, and as such it is a very attractive target for obesity reduction. Because it is a mitochondrial sirtuin, SirT3’s activity leads to decreased acetylation of mitochondrial proteins, which invariably results in increased activity (Shi et al., 2005). We have recently shown that the isoquinoline alkaloid berberine is a potent inducer of SirT3’s activity in high fatefed rats, which at least partially explains this compound’s potent anti-obesity effects (Teodoro et al., 2013). Because SirT1 is a NADþ-dependent deacetylase, its activity is dependent on the cell’s reductive status, and a high nutrient ambient leads to decreased SirT1 activity, where PGC1a remains acetylated and its activity diminished (Canto´ and Auwerx, 2009). Another metabolic sensor, the AMP-activated protein kinase (AMPK), is activated in the presence of low energy levels, i.e., when the ATP levels are low (or, more appropriately, when AMP levels are high). It phosphorylates PGC1a, activating it to produce more mitochondria to try to elevate ATP levels (Canto´ and Auwerx, 2009). As such, these sensors’ activities on PGC1a were designed to counter situations of low energy stress and are completely shut down in obesity and diabetes. Consequently, the artificial induction of their activation can be considered a potential and extremely attractive therapeutic strategy, especially considering that oxidative phosphorylation inhibition is a hallmark of HFD and diabetic animals. AMPK is an important metabolic sensor and regulator, being involved in, among other effects, glucose uptake, lipidic b-oxidation, and mitochondrial biogenesis. Its effects are present on several organs, from the liver to the brain, from WAT and BAT to skeletal muscle, i.e., all metabolic-relevant tissues (Winder et al., 2000). Because AMP activates AMPK, a rise in this adenosine nucleotide (with concomitant decrease in ATP) signals the cell to begin substrate oxidation processes to generate ATP. As paralleled by NADþ and SirT1, this (and subsequent downstream effects) can be explored (and has been extensively studied) for obesity management. Although SirT1 deacetylates proteins and histones, AMPK phosphorylates and alters proteins’ activity (either increasing or decreasing) (Hardie et al., 2012). AMPK induces GluT1 activation and GluT4 migration to the cellular membrane and thus increased

glucose uptake and oxidation (Barnes et al., 2002; Pehmøller et al., 2009). AMPK also mediates fatty acid uptake in cardiac cells (Habets et al., 2009), while improving their uptake and oxidation mainly by the inhibition of acetyl-CoA carboxylase and thus increasing mitochondrial import of fatty acids (Merrill et al., 1997) and by increasing the glycolytic rate (Marsin et al., 2002). Another key effect of AMPK is on mitochondrial biogenesis for, unsurprisingly, AMPK phosphorylates and activates PGC1a, thus increasing mitochondrial content, particularly in skeletal muscle (Winder et al., 2000). Finally, AMPK can also activate (and be activated by) SirT1, by increasing cellular NADþ levels (Canto´ et al., 2010). As with sirtuins, these are just some effects of AMPK on metabolism, for it is also involved in many other biological functions. For further reading, we refer the reader to Hardie et al. (2012). Oxidative stress also plays a major role in mitochondrial dysfunction in high-energy situations. In fact, as noted before, increased ROS generation is common in high-energy situations. The increased ROS generation, along with mitochondrial damage, also causes the activation of inflammatory pathways (as the c-Jun N-terminal kinase [JNK] and mitogen-activated protein kinase [MAPK]). These cause the inactivation of the insulin signaling pathway and loss of GluT4 (the insulin-sensitive glucose transporter) translocation to the cellular membrane (Qatanani and Lazar, 2007), increasing insulin resistance and thus exacerbating the problem. Also, because of the high energy levels, it comes as no surprise that the expression of proteins involved in lipid handling and mitochondrial lipid b-oxidation is diminished (Schreurs et al., 2010). Furthermore, ROS contribute to diminish glycolytic rates, as it is known that ROS inhibit the key glycolytic enzyme GAPDH (Du et al., 2000), which appears to be a self-defense mechanism against glucose damage (Rolo and Palmeira, 2006). Also, the persistent excess of nutrients leads to the maintenance of said inhibition and worsening of the situation. All of this contributes to increased lipid deposition inside cells, which affects not just mitochondrial function, but also the entire cell. Increasing the number of mitochondria is an attractive strategy for it leads to more units to carry the load of more nutrients, because not only would ROS generation be attenuated but also more antioxidant defenses would also be present. On the other hand, mildly uncoupling mitochondria leads to decreased ROS generation by the caloric dissipation of the electrochemical protonic gradient (Korshunov et al., 1997; Skulachev, 1998). There is already much work being conducted on both perspectives, both yielding very promising results (for further reading, please refer to Ren et al., 2010; Wu et al., 2013).

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MITOCHONDRIAL DYSFUNCTION IN ISCHEMIA/REPERFUSION

In terms of increasing mitochondrial numbers, there has been an enormous push toward research on brown adipose tissue since its recent discovery in human adults. Brown adipose tissue (BAT) evolved into the main source of heat generation in small mammals by having a high content of UCP1-expressing mitochondria (UCP1 can be and is considered a hallmark of BAT) (Cannon and Nedergaard, 2004). When activated, BAT generates a high metabolic rate, sustained by a rather large rate of substrate oxidation, which is obtained both from its lipid droplets and from circulation (Bartelt et al., 2011). As such, overactivation of BAT is, theoretically, a highly effective therapeutic strategy for obesity. In fact, we have recently shown just that, with the use of the bile acid chenodeoxycholic acid (CDCA) an obese phenotype can be normalized by elevating BAT UCP1 activity (Teodoro et al., 2014). It was thought that BAT was not present in adult humans, voiding such therapeutic approaches, but, as noticed before, it has been shown otherwise (Whittle, 2012). However, Vosselman et al. (2012) demonstrated that overstimulation of BAT in adult humans was hardly a valid strategy. As such, thermogenic therapy for obesity in adult humans could only be a failed idea, if not for the fact that adipocytes are highly plastic cells. This means that, given the right stimuli, white adipocytes can be, to some extent, converted into brown-like cells, the so-called “brite” or beige adipocytes (the opposite, i.e., the conversion of brown into white is also possible). As such, the next “big thing” in metabolic research is the conversion of white into brown adipocytes thus creating elevated basal metabolic rates, burning more fuel, and decreasing obesity. Most therapeutic strategies already studied and reported, which reduce adiposity in white adipocytes, produce metabolic alterations that are common to what is described to happen in “brite” inductiondi.e., the activation of PPARa and induction of lipolysis, increased leptin release, and induction of mitochondrial biogenesis (Flachs et al., 2013). These alterations are usually associated with UCP1 induction and nonshivering thermogenesis. Curiously, UCP1-null mice are obesityresistant when exposed to cold, but not at thermoneutrality (Anunciado-Koza et al., 2008). To make matters worse, the work by Nedergaard and Cannon (2013) skillfully argues that, despite the huge increase in UCP1 expression in WAT (arising from virtually zero), the overall contribution of these newly formed “brite” cells to the body’s basal metabolic rate is negligible at best. As such, the authors propose that some other mechanism is responsible for the effects demonstrated in other works. Flachs et al. (2011) suggest that n-3 PUFA antiobesogenic effects are not UCP1-dependent, which is also the case when combined with calorie restriction, but they are caused by increased cycling of triglyceride/free fatty acid cycle (TG/FFA cycle), a so-called

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metabolic futile cycle, for it consumes energy while not causing the generation of products. This cycle could be behind many anti-obesogenic effects of countless compounds, whose effects are clear, but whose mechanisms are not. Research into these metabolic pathways will probably become a hot topic for obesity research in the near future. We have shed some light into this matter, by demonstrating once more using CDCA that there is more than just thermogenic dissipation behind bile acids’ anti-obesity effects, in particular, an acceleration of metabolic functions (Teodoro et al., 2016).

MITOCHONDRIAL DYSFUNCTION IN ISCHEMIA/REPERFUSION Ischemia/reperfusion (I/R) injury is a phenomenon whereby damage to a hypoxic organ is accentuated following the return of the oxygen supply, and it has been recognized as a clinically important pathological disorder. I/R may occur in many clinical situations such as transplantation, resection, trauma, shock, hemorrhage, and thermal injury. The mitochondrial function is impaired in I/R settings, leading to an alteration of energy metabolism. Ischemia leads to the cessation of oxidative phosphorylation, which causes tissue ATP and creatine phosphate concentrations to decrease with a simultaneous increase in ADP, AMP, and inorganic phosphate (Pi) concentrations. During ischemia, anaerobic glycolysis and ATP degradation produce Hþ-maintaining mitochondrial membrane potential. As maintenance of ion gradients across the plasma membrane and between cellular compartments depends on ATP-driven reactions, metabolic disruption by injurious stresses may rapidly perturb cellular ion homeostasis. During oxygen deprivation the intracellular Hþ, Naþ, and Ca2þ levels are elevated, inducing osmotic stress and causing mitochondrial damage. Intracellular Hþ accumulation activates the Naþ/Hþ exchanger, leading to Naþ influx. Naþ efflux is attenuated because the Naþ/Kþ-ATPase is inhibited during ischemia. Therefore, Naþ/Hþ exchange activity leads to increasing intracellular Naþ (Inserte et al., 2002, 2006; Murphy and Steenbergen, 2008). This augmentation of intracellular Naþ during ischemia is accompanied by an increase in intracellular Ca2þ through reverse mode of the Naþ/Ca2þ exchanger. Although Naþ overload stimulates Ca2þ influx by the Naþ/Ca2þ exchanger and depletion of ATP reduces Ca2þ uptake by the endoplasmic reticulum, the Ca2þ level is maintained as modest during ischemia because acidosis inhibits the Naþ/Ca2þ exchanger, and cytosolic Ca2þ is taken up by the mitochondria as long as its membrane potential is maintained. Influx of extracellular Ca2þ is responsible

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for irreversible cell injury as shown by studies in which removal of extracellular Ca2þ protects against various hepatotoxicants (Schanne et al., 1979; Farber, 1982). ROS generation plays a major role in damaging the organ during ischemia and sensitizing it to reperfusion. The source of the ROS is uncertain, perhaps involving complexes I and III of the ETC of mitochondria, or perhaps xanthine/xanthine oxidase (X/XOD) acting on xanthine formed from the degradation of adenosine (AMP is slowly converted into adenosine and then inosine and xanthine through a purine degradation pathway). There is a gradual decline in cellular integrity as a consequence of combinative action of ATP depletion, elevated intracellular Ca2þ, and ROS. Thus, the ATP-dependent repair processes are incapable of operating. Maintenance of mitochondrial integrity is a critical determinant of cell outcome. As such, if mitochondria remain sufficiently intact to generate ATP after short periods of ischemia, tissue damage is reversible and can be repaired. But if the ischemia is more aggressive then recovery is not possible. The ATP restored during reperfusion will exacerbate the damage to the organ due to metabolic disorders that accumulate during ischemia leading to cell death. The increased Ca2þ and bursts of ROS generation are characteristics of reperfusion. Probably the majority of ROS is formed by uncoupled mitochondria, mainly from mitochondrial complexes I and III of the ETC (Jaeschke and Mitchell, 1989; Turrens, 2003). When the respiratory chain is inhibited by absence of oxygen and then reexposed to oxygen, ubiquinone can become partially reduced to ubisemiquinone. It can then react with oxygen to generate superoxide that is reduced to hydrogen peroxide by superoxide dismutase. Hydrogen peroxide is removed by glutathione peroxidase or catalase, but if ferrous ions (or other transition metals such as copper) are present it will form the highly reactive hydroxyl radical through a Fenton reaction (Becker, 2004). In fact, mitochondrial lipids and proteins that are damaged during ischemia favor ROS generation during reperfusion (Inserte et al., 2002). ROS cause peroxidation of cardiolipin of the inner mitochondrial membrane, impairing electron flow through the ETC (Petrosillo et al., 2003; Paradies et al., 2004). Moreover, lipid peroxidation causes the release of reactive aldehydes such as 4-hydroxynonenal that alters membrane proteins (Echtay et al., 2003). ROS also have direct effects on several respiratory chain components and can cause inhibition of the ATP synthase and ANT (adenine nucleotide translocase). There is depletion in superoxide dismutase, glutathione peroxidase, and glutathione during reperfusion that enhances oxidative stress. Mitochondria are the major target of ROS and Ca2þ overload. These agents are potent inducers of the MPT,

resulting in mitochondrial-initiated cell death. A major consequence of MPT induction is inhibition of oxidative phosphorylation, which when unrestrained will lead to necrotic cell death. The permeability transition has also been pointed to as being involved in apoptosis, through the release of proapoptotic factors, such as cytochrome c, and other apoptosis-inducing factors into the cytosol (Forbes et al., 2001; Murata et al., 2001). In response to proapoptotic signals, Bax, a proapoptotic member of the Bcl-2 family, is translocated to the mitochondria and can form channels that allow the release of cytochrome c from the mitochondrial intermembrane space (Borutaite and Brown, 2003). In conditions of ATP depletion, apoptosis can deviate to necrosis (necroapoptosis). Changes in mitochondrial morphology achieved by fission and fusion may play an important role as a determinant of cell viability. It is important to understand the molecular mechanisms of mitochondrial dynamics and their relationship with ischemia-reperfusion injury. Given the role of mitochondria in ischemia/reperfusion injury, strategies have been developed that focus on maintaining mitochondrial function and consequently reducing the damage. Perfusion with GSK-3b inhibitors reduces cell death induced by I/R (Tong et al., 2002; Gross et al., 2004; Pagel et al., 2006; Gomez et al., 2008). It is thought that the mechanism of protection elicited by GSK-3b inhibition is related to modulation of MPT, by interaction between GSK-3b and components of the MPT process (Juhaszova et al., 2008). Phospho-GSK-3b can bind to the ANT, voltagedependent anion channel (VDAC), or Cyclophilin D (CypD) (Pastorino et al., 2005; Nishihara et al., 2007). Pretreatment with indirubin-30 -oxime (an inhibitor of GSK-3b) in conditions of hepatic I/R protects the liver by maintaining mitochondrial calcium homeostasis, thus preserving mitochondrial function and hepatic energetic balance (Varela et al., 2010). GSK-3b inactivation by indirubin-30 -oxime acts as pharmacological preconditioning, modulating the susceptibility to MPT induction and preserving mitochondrial function after I/R. The suppression of the ANT-CypD interaction may contribute to the elevation of the threshold for MPT induction. CypD null mice mitochondria have been demonstrated to have higher Ca2þ buffering capacity, demonstrating a desensitization of these mitochondria to Ca2þ-induced MPT (Baines et al., 2005). Recently, a relationship was established between SirT3 and CypD: SirT3 deacetylates and inactivates CypD causing its dissociation from the ANT (Shulga et al., 2010). The decrease in SirT3 activity leads to increased activation of the MPT in response to Ca2þ increases, cardiac stress, and aging, resulting in a decline in cardiac function (Hafner et al., 2010). This ability to suppress MPT formation indicates SirT3 as a potential target for new drugs that protect against I/R.

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MITOCHONDRIAL DYSFUNCTION IN ISCHEMIA/REPERFUSION

Mitochondrial K-ATP channels are normally closed in vivo because of the inhibitory concentrations of ATP and ADP. Mitochondrial K-ATPedependent matrix alkalization, preservation of mitochondrial volume, and the structure of the intermembrane space, as well as MPT inhibition, are involved in a preconditioning protective action (Andrukhiv et al., 2006; Costa et al., 2006; Costa and Garlid, 2008). Diazoxide is a selective mitochondrial K-ATP channel agonist that was previously shown to decrease I/R injury induced by orthotopic liver transplantation (Huet et al., 2004). The protective effects of diazoxide against hepatic I/R were dependent on Bcl-2 expression and also with the inhibition of mitochondrial cytochrome c release, being abolished by siRNA knockdown of Bcl-2 (Wu et al., 2010b). We have recently contributed to the demonstration of the essential role of mitochondrial function preservation in an I/R setting (Alexandrino et al., 2016) (Fig. 55.2). The events of ischemia/reperfusion lead to a number of cellular alterations, with particular relevance for mitochondrial function. During ischemia, restriction of blood flow limits access to nutrients, ions and, most relevantly, oxygen. Because ATP requirements are maintained (and, in some cases, elevated), ATP generation drains the mitochondrial membrane potential. Eventually, the cell has to resort to anaerobic generation

[O 2]

of ATP through glycolysis, which results in the accumulation of lactate and concomitant decrease in pH. Furthermore, ion exchanges are altered leading to intracellular Naþ accumulation and Ca2þ overload (see full text for further details). Accompanying the decrease in mitochondrial function, there is a mild increase in ROS generation. If the ischemic event is prolonged and if the cell was not prepared for it (for example, by preconditioning it with pharmacological agents or with short, repetitive ischemia/reperfusion events), during reperfusion, the restoration of blood flow and particularly of oxygen restores mitochondrial activity but in a totally different setting. Mitochondrial environment and function are compromised because of alterations suffered during ischemia, and ROS generation is highly exacerbated, with resulting damage to mitochondrial components such as the members of the respiratory chain and cardiolipin, heightening the problem. All this leads to the induction of the mitochondrial permeability pore, with concomitant release of cytochrome c and other proapoptotic factors, which might lead to cell death. Various therapeutic agents have already been shown to be modulators of mitochondrial function and normalizers during ischemia/reperfusion. Of note, berberine leads to an overactivation of SirT3 and GSK-3b inhibitors such as indirubin-30 -oxime (see text for further details).

[O2] Berberine

Anaerobic glycolysis

ATP degrada on H+

GSK-3β inhibitors

Re-energiza on of mitochondria

pH restora on

Lowering pH Cell Death

Na+

GSK-3β Sirt3

Na+

ETC

Ca2+

ETC

ROS

Mild ROS amount

GPx Catalase

H+ Oxidazed CL

CL

Na+ ROS & Ca2+ overload

Na+ Ca2+

∆Ψm

∆Ψm

FIGURE 55.2 Role of mitochondria in Ischemia/Reperfusion injury. DJm, mitochondrial membrane potential; CL, cardiolipin; ETC, electronic transport chain; GPx, glutathione peroxidase; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species.

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55. BIOMARKERS OF MITOCHONDRIAL DYSFUNCTION AND TOXICITY

MITOCHONDRIAL DYSFUNCTION IN CANCER As noted before, mitochondria are responsible for a wide array of reactions and phenomena in the cell. Energy (ATP) production, redox status maintenance, ROS generation, Ca2þ storage, and apoptosis control, to name a few, are all dependent on and/or take place in mitochondria. As such, it comes as no surprise to find that mitochondrial alterations and/or deregulation are involved in cancer development. In fact, alterations in biosynthetic pathways, cell signaling, or DNA replication can shift a cell from a quiescent to a proliferative status (Wallace, 2012). The idea of mitochondrial dysfunction in cancer is rather ancient, dating back to the description of the Warburg effect, meaning the realization that cancerous cells have increased lactic acid production in the presence of oxygen, the so-called aerobic glycolysis. This led to the idea that cancer cells have damaged mitochondria because pyruvate is converted into lactate, and glycolysis is the main source of ATP for the cancerous cell. This is further supported by the notion that the interior of tumors is a highly anoxic area, elevating the need for glycolytic-derived ATP and downplaying the need for a functional mitochondrial population. All these factors would come into play in creating a highly proliferative, malignant cell. As discussed below, a healthy mitochondrial population is as vital to a normal cell as it is to a cancerous one. It is noteworthy, however, that it is true that mtDNA is highly mutated in cancer cells and these cells have a higher glycolytic rate than do normal cells. It is the fact that mitochondria are not essential to the cancerous cell that is in dispute. One way by which we can demonstrate that mitochondria are essential to cancerous cells comes from studies where the mtDNA was removed by ethidium bromide exposure, generating the so-called r0 cells. These cells present a lower metabolic rate, resulting in decreased growth and tumor formation (Cavalli et al., 1997; Weinberg et al., 2010). In his seminal review, Wallace (2012) points to Tasmanian devils’ and dogs’ transmissible tumors, which cause mtDNA decay and which would have disappeared long ago, if not for periodic uptake of normal mtDNA from host cells. Both somatic and germline mtDNA mutations have been reported in almost all cases of tumors and cancers. Despite the fact that some of these alterations could be considered normal heterogeny of the population, there are no doubts about the direct correlation of some of these mutations with a cancerous phenotype. Consequently, almost all cancerous cells demonstrate impaired OXPHOS activity, when compared with normal cells. As such, these alterations can be beneficial to the cancer cell,

by promoting neoplastic alterations and allowing the cancer cell to adapt to a wide array of metabolic environments (Brandon et al., 2006). In fact, several mtDNA mutations have been demonstrated to be positively selected in cancer cells, in contrast to normal cells (Gasparre et al., 2008). But the presence of a mutation that will affect mtDNA and thus lead to a cancerous phenotype appears to not even be necessary, for it has been shown that the nuclear-encoded SUV3 RNA helicase is required for correct mitochondrial function and biogenesis. In fact, SUV3þ/þ can present the same altered mitochondrial phenotype of a heterozygous or double recessive individual, because of maternal inheritance of altered mitochondria (Chen et al., 2012). But if cancerous cells do indeed present mitochondrial alterations and impaired OXPHOS activity, then how is it possible that functional mitochondria are needed for the viability of said cells? For one, mitochondrial activity can be altered by more than just mtDNA mutations. In fact, various nDNA mutations for mitochondrial proteins have been reported in various types of cancers. For example, mutations in the complex II proteins have been shown to lead to increased ROS generation, decreased succinate consumption, and decreased respiratory rates and thus a shift toward glycolysis, in a mechanism that appears to involve the stabilization of the hypoxia-inducible factor 1a, HIF1a (Wallace and Fan, 2010; Kurelac et al., 2011). In the same fashion, fumarate hydratase, an enzyme of the Krebs cycle, has also been shown to be mutated in various cancers, leading to increased succinate levels and thus the shift toward increased glycolytic metabolism but, apparently, without the involvement of HIF4a (Adam et al., 2011; Frezza et al., 2011). There are many other reported mutations in other mitochondrial proteins, for example isocitrate dehydrogenase (Ward et al., 2012), and complexes I, III, IV, and V of the respiratory chain (Wallace, 2012). Mitochondria produce ROS as a by-product of their activity. It is estimated that roughly 5% of all oxygen consumed in a cell is not converted into water but rather into a reactive species. Although a by-product, it has been recently shown that ROS have a signaling activity (Lander, 1997; Devasagayam et al., 2004), resulting in their production being not only required but also needed (within certain limits). As such, a complex (and, for most cases, effective) antioxidant system is in place to reduce them and thus neutralize their activity. However, mitochondrial defects, metabolic imbalances, mutations, and lack of mtDNA histones all lead to increased mtDNA mutations and thus increased cancerous potential. ROS can be one of the most important causal factors for said mutations. In fact, it has been demonstrated that ROS generation is augmented by

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MITOCHONDRIAL DYSFUNCTION IN CANCER

inhibition of the OXPHOS, in particular of the ATP synthase (Sa´nchez-Cenizo et al., 2010). In addition to contributing to increased ROS generation, this blockade of OXPHOS also leads to the already discussed metabolic shift toward glycolysis. We have previously mentioned that the shift toward a greater dependence on glycolytic-derived ATP is a standard condition in the progression from a normal to a cancerous cell. This shift typically involves the overactivation of the PI3KeAkt signaling pathway (Jones and Thompson, 2009), which leads to increased expression of glucose transporters, glycolytic and lipogenic enzymes, and activation of the mechanistic target of rapamycin, mTOR (DeBerardinis et al., 2008), resulting in elevated rates of glycolysis and lactate generation. This pathway also inhibits fatty acid oxidation and prevents carbon flow toward the Krebs cycle (Gatenby and Gillies, 2004; DeBerardinis et al., 2008). Furthermore, by inhibition of the master regulator of mitochondrial biogenesis, PGC1a, this pathway leads to decreased mitochondrial respiratory activity and content, of both respiratory chain units and antioxidant defenses (Daitoku et al., 2003). As mentioned before, HIF1a stabilization is a recurring phenomenon in cancer cells, so it comes as no surprise to realize that HIF1a can impair mitochondrial biogenesis (Zhang et al., 2007). But why does the cancerous cell go to such an extent as to possibly limit its own ATP production? The answer is rather simple: the downplay of mitochondrial bioenergetics is a side effect of the need for the activation of glycolysis-parallel anabolic pathways. By increasing glycolysis, more carbon is shunted toward, for example, the pentose phosphate pathway for nucleotide synthesis and NADPH generation to combat oxidative stress (Gru¨ning et al., 2011). Also, the increased generation of glycerol-3-phosphate in glycolysis leads to heightened lipogenesis (Esechie and Du, 2009), which requires mitochondrial-derived acetyl-CoA, supplied by a complex process that involves the aggressive oncogene Myc, in an anaplerotic refill of Krebs cycle intermediaries (DeBerardinis et al., 2007), making cancer cells dependent on glutamine for their survival (Wise et al., 2008). The antitumor p53 protein provides another proof of mitochondrial dependence of cancer cells. p53 inhibits glycolysis and diverts carbon toward the pentose phosphate pathway, leading to increased NADPH generation; in addition, its activation by telomere shortening prevents PGC1a-mediated mitochondrial biogenesis, thus leading to increased ROS generation and cellular senescence (Sahin and Depinho, 2010). As such, it is clear that, for a cancerous cell, a viable, efficient mitochondrial population is a vital requirement (Vander Heiden et al., 2009). There are many other ways by which mitochondrial function is linked with cancer (masterfully reviewed in

991

Wallace, 2012). If mitochondria are so vital for the cancer cell, then it is only reasonable to assume that targeting mitochondria to try to treat cancer is a viable therapeutic strategy. In fact, various works (Fulda et al., 2010; Hockenbery, 2010; Wenner, 2012) have focused on the subject. Therefore, we will briefly approach the thematic in these works. Cancer trademarks, such as immortal potential, unresponsiveness to growth arrest signaling, increased anabolic metabolism, and decreased apoptosis and autophagy, have already been linked to mitochondria (Galluzzi et al., 2010). Despite the already discussed dependence on mitochondrial activity, cancer cells have structurally and functionally different mitochondria when compared with normal cells (ModicaNapolitano and Singh, 2004). One way to target mitochondria is by targeting its membrane potential (DJm). Specific ANT ligands can lead to the induction of the MPT (Lehenkari et al., 2002; Don et al., 2003; Oudard et al., 2003) and could be a valid strategy because cancer cells should be more susceptible owing to having higher metabolic rates and higher Ca2þ loads. Also, the peripheral benzodiazepine receptor (PBR) is thought to be a component of the MPT (as well as ANT) and binds to the voltage-dependent anion channel (VDAC) and prevents MPT induction. Thus, it comes as no surprise that PBR is typically overexpressed in various cancers, as it blocks the antiapoptotic effect of the Bcl-2 protein family. As such, PBR ligands have shown antitumor activity (Decaudin et al., 2002). These are but a few examples of how reducing DJm and inducing apoptosis can be considered a valid and promising anticancer strategy. In fact, various compounds are already in test that target these described mechanisms. It is evident that a compound or therapeutic strategy that would increase ROS generation or inhibit antioxidant defenses in cancer cells could be immensely helpful. Along these lines, there are already some promising results with ROS inducers (Sarin et al., 2006; Bey et al., 2007; Mehta et al., 2009) and antioxidant defense compounds (Alexandre et al., 2006; Trachootham et al., 2006; Dragovich et al., 2007), to name a few. In the mitochondrial intermembrane space reside various proapoptotic factors (e.g., cytochrome c, cyt c), and their release is a common phenomenon in mitochondrial-dependent apoptosis, a phenomenon that usually involves the proapoptotic proteins BAX and BAK (Chipuk et al., 2006). As such, the modulation of the activities of the pro- and antiapoptotic family of proteins (for example, the Bcl-2 family) can provide interesting therapeutic approaches. The compounds ABT-737 (Oltersdorf et al., 2005) and ABT-263 (Tse et al., 2008) are just two examples. Of course, targeting mitochondrial metabolic activity is also a strong possibility. Inhibition of the pyruvate

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55. BIOMARKERS OF MITOCHONDRIAL DYSFUNCTION AND TOXICITY

dehydrogenase kinase (which blocks PD activity of converting pyruvate into acetyl-CoA) by dichloroacetate leads to increased ROS generation in cancerous, but not in normal cells (Bonnet et al., 2007). Also, inhibition of the expression of lactate dehydrogenase A has shown promising results, by impeding the conversion of pyruvate into lactate (Fantin et al., 2006). Pyruvate is also actively sent toward lipid synthesis, so the block of the key enzyme ATP citrate lyase has also shown promising results (Hatzivassiliou et al., 2005). These are only a few examples of how targeting mitochondrial metabolism is a valid therapeutic strategy to combat cancer. There are many other compounds and therapeutic strategies and targets being currently tested against cancer, many of them also involving mitochondria. Therefore, it would be no surprise if the cure for cancer came from a strategy that involved mitochondria.

CONCLUDING REMARKS AND FUTURE DIRECTIONS Considering that mitochondria play an essential role in cellular homeostasis and signaling processes, identifying biomarkers of mitochondrial dysfunction and toxicity is of fundamental relevance. Research into these mitochondrial targets is at present a hot topic in several diseases (diabetes, obesity, cancer, etc., to name just a few examples) and drug-induced toxicity. An improved understanding of the changes that occur at the mitochondrial level is essential to discover new therapeutic targets for mitochondria-related diseases and toxicity exposure.

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