An homeostatic balance exists in physiological conditions between the formation of reactive oxygen species (ROS) and their elimination by endogenous antioxidants . As a consequence of elevated production of ROS occurs oxidative stress which is involved in a high number of human diseases. These ROS include: superoxide (O2.-), hydrogen peroxide (H2O2), hydroxyl radicals (.HO), peroxynitrite (ONOO-) and others . Endothelial dysfunction is also correlated with oxidative stress, and both are related to risk factors for several diseases, as well as cardiovascular events and negative long-term outcome .
The mitochondrion is the main source of ROS. Antioxidants can exert a specific action on the mitochondrial respiratory chain, which constitutes an important mechanism of protection. ROS cause non-specific damage to lipids, proteins and DNA, leading to alteration or loss of cellular function. As a consequence, mitochondria are more vulnerable to oxidative damage than other cellular organelles, and accumulate oxidative damage more rapidly than the rest of the cell .
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Knowledge of mitochondria's crucial role in the life and death of cells has created a need to develop cytoprotective agents that are targeted to mitochondria. Physiologically, mitochondria perform a different key cellular regulatory processes and use approximately 90% of the O2 consumed for oxidative phosphorylation. Mitochondrial oxidative damage and impairment contribute to a number of cell pathologies. Studies in different models have shown that the harmful effects of ROS are offset by antioxidants such as N-acetylcysteine, ubiquinol and ï¡-tocopherol, which reduce mitochondrial oxidative damage [5-7]. Due that these compounds do not accumulate property in the mitochondria, the effectiveness of these compounds is limited , and for this reason there are research efforts to develop mitochondria-targeted molecules. In the bibliography, it has been described that triphenyl-phosphonium-(TPP)-based antioxidants and amino acids and peptides, which are cell-permeable, having high viability, reducing intracellular ROS and preventing cell death mitochondria can protect against oxidative stress [9-11].
OXIDATIVE STRESS: ROS AND REACTIVE NITROGEN SPECIES (RNS)
The role of ROS-mediated damage in many major disorders it is very important to know [10-11]. Oxygen is a major source of ROS and the most ubiquitous of all biologically essential chemical species. Oxidative stress consists on an imbalance between oxidant production and antioxidant defences , and is associated with many of the risk factors implicated in the pathophysiology of multiple diseases such as atherosclerosis, diabetes, hypercholesterolaemia or ageing .
ROS are generated by biochemical reactions in the cell, and balanced levels depend on the physiological and pathophysiological states of the organism. In fact, under physiological conditions, a homeostatic balance exits between the formation of ROS and their elimination by endogenous antioxidant scavenging compounds and enzymes . Oxidative stress occurs when this balance is disrupted by excessive production of ROS, including O2ï‚· ï€, H2O2 and ï‚·OH, and/or inadequate antioxidant defences , including ascorbic acid (AA), ï¡-tocopherol, GSH, SOD and CAT. In this way, ROS are important secondary messengers that are generated in response to different forms of environmental stress, and changes in their intracellular levels can activate signal transduction pathways and influence the interaction of cells with their environment.
ROS and reactive nitrogen species (RNS) are closely linked to the disease process, and can potentially damage all cellular components. Structure alteration, biomolecule fragmentation and oxidation of side chains are trade-offs of cellular energy production. Deficient scavenging of ROS and RNS particularly affects the mitochondrial lipid cardiolipin (CL), triggers the release of mitochondrial cytochrome c and activates the intrinsic death pathway. Due to the active redox environment and the excess of NADH and ATP at the inner mitochondrial membrane, a broad range of agents, including electron acceptors, electron donors and hydride acceptors, can be used to exert an influence on biochemical pathways. The therapeutic value of these agents lies in their enrichment of selective redox modulators at target sites.
ROS are generated by biochemical reactions in the cell. Leakage of electrons from the mitochondrial electron transport chain is a significant source of mitochondrial ROS, particularly O2ï‚·ï€ , which is formed spontaneously in the electron-rich aerobic environment of the inner mitochondrial membrane within the respiratory chain. It is converted to H2O2 by the enzyme SOD, and the tricarboxilic acid cycle enzymes ï¡-ketoglutarate dehydrogenase and pyruvate dehydrogenase complex generate both O2ï‚·ï€ and H2O2 . H2O2 is not itself a free radical, but is nonetheless important due to its ability to penetrate biological membranes and is a biological marker of oxidative stress. Excess H2O2 is normally converted to water through a harmless action exerted by GSH peroxidase (GPx), CAT and other peroxidases. In the presence of metal ions, ï‚·OH can be formed by the reaction of O2ï‚·ï€ with H2O2. ï‚·OH is much more reactive than O2ï‚· ï€. Iron-catalysed ï‚·OH generation requires iron to be in its reduced, ferrous form (Fe2+), whereas most of the iron in cells and plasma is found in the oxidized form (Fe3+). In addition to its involvement with H2O2 in ï‚·OH formation, O2ï‚· ï€ reduces Fe3+ to Fe2+, thereby further promoting ï‚·OH production. Under normal physiological conditions, the majority of ROS are formed during cellular respiration and by activated phagocytic cells involved in the inflammatory response, such as neutrophils or macrophages.
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In diabetes, ROS are generated by several potential sources, endothelial cells, phagocytic cells of the immune system (the respiratory burst), nitric oxide synthase, release of iron and copper ions, metalloproteins, vascular damage caused by ischemia reperfusion, the mitochondrial respiratory electron transport chain, xanthine oxidase (XO) activation, the respiratory burst associated with immune cell activation, and arachidonic acid metabolism. NAD(P)H oxidase, which catalyzes the production of O2ï‚· ï€ by one-electron reduction of O2 using NADPH or NADH as the electron donor, exists both in phagocytes and in non-phagocytes, including fibroblasts, chondrocytes and mesangial, microglial, epithelial, endothelial and vascular smooth muscle cells.  This particular enzyme displays a key role in the development of diabetes.
Importantly, as oxidants produced by immune cells, ROS and RNS have a dual function. On the one hand, they function as potent antimicrobial agents by killing microbial pathogens. On the other hand, they constitute signalling molecules that regulate diverse physiological signalling pathways in neutrophils. In the latter role, ROS and RNS act as modulators of protein and lipid kinases and phosphatases, membrane receptors, ion channels and transcription factors including NF-ï«B. This latter role is exercised through the regulation of key cytokines and chemokines, which modulates the inflammatory response, during which ROS and RNS, in turn, modulate phagocytosis, secretion, gene expression and apoptosis. Under pathological circumstances, excess production of ROS can interfere vicinal cells such as those found in the endothelium or epithelium, thereby contributing to inflammatory tissue injury . In this context, the typical behaviour of these cells in a state of oxidative stress implies changes in different immune functions, such as an increase in adherence and phagocytosis and a decrease in chemotaxis .
Three simple oxides of nitrogen are the focus of current biomedical interest: NO, nitrous oxide (N2O) and nitrogen dioxide (NO2). NO2 is an environmental pollutant that seems to be produced in vivo in response to reactions of NO and is an initiator of lipid peroxidation. NO reacts poorly with most molecules in the human body (non-radicals), but as a free radical it reacts extremely rapidly with other ROS such as O2ï‚·ï€, with amino acid radicals, and with certain transition metal ions. The reaction between NO and O2ï‚· ï€ produces peroxynitrite (ONOO-) , which is itself a powerful oxidant that decomposes and yields further oxidants with the chemical reactivity of NO2, ï‚·OH and NO2+. Diminished availability of NO and increased ROS formation may constitute key events in the pathology of atherosclerosis  and as a consequence of cardiovascular diseases.
The activation of monocytes, macrophages and endothelial cells results in the expression of iNOS, and consequently promotes the transformation of L-arginine into NO, which can combine with O2ï‚·ï€ to form ONOO-. NO stimulates H2O2 and O2ï‚·ï€ production by the mitochondria , possibly by inhibiting cytochrome c oxidase (COX), thus stimulating the leakage of electrons from the respiratory chain. H2O2, in turn, is involved in the upregulation of iNOS expression via NF-ï«B activation. In tissue injury, inflammatory reactions play an important role that is mediated by adhesion and migration of leukocytes through the endothelium, generation of ROS and RNS, and the release of several proinflammatory cytokines by monocytes/macrophages. Moreover, local generation of RNS contributes to tissue injury.
OXIDATIVE STRESS AND MITOCHONDRIA
There is a growing body of evidence that implicates the mitochondria in cellular signalling pathways and these actions modulate intracellular calcium stores and production of ROS. Furthermore, the importance of mitochondria has renewing highlighting the interaction of NO with mitochondrial functions such as respiration and biogenesis. Given these important roles, alterations in mitochondrial function are thought to be key to the development of human disease [19-20].
Mitochondrial diseases have been related to mitochondrial respiratory-chain deficiencies associated with mutations of mitochondrial DNA, and inherited dysfunction of the mitochondrial oxidative phosphorylation system is increasingly recognized in humans [21-22]. Most of the protein subunits of the electron transport chain (ETC) are encoded by nuclear genes, while only 13 essential subunits are encoded by mitochondrial DNA (mtDNA). A series of nuclear and mtDNA mutations have been identified as the cause of defects and isolated disorders of individual oxidative phosphorylation enzymes, including mitochondrial ATP synthase in patients with cardiovascular-metabolic diseases . The diseases attributed to familial mtDNA deletions and mutations are less common than those related to nuclear DNA defects, which may be a result of mitochondria containing several copies of their genomes, as in the continuous fusion of mitochondria, modified genes are mixed with normal genes. This particular process is very important in human diseases.
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The discovery that acute and chronic stress in cells leads to structural and functional impairments of mitochondria has caused the role of mitochondria in disease etiology to be redefined , and is of some relevance to cancer. Mitochondrial dysfunction triggers signalling cascades for cell necrosis and apoptosis, and leads to organ failure and the development of disease. The list of mitochondria-related conditions diseases is growing rapidly and includes cancer, heart failure, diabetes, obesity, stroke, neurodegenerative diseases and aging, which are characterised by disturbances of mitochondrial Ca2+, ATP or ROS metabolism [24-25]. Mitochondrial dysfunction has been shown to contribute to the progression of neurodegenerative diseases, such as Parkinson's, and to stroke , insulin resistance  and nitroglycerine tolerance .
As mitochondria are the major site of generation of cellular oxidative stress and play a key role in mediating programmed cell death (apoptosis), it is feasible that damage to mtDNA contributes in a significant way to human ageing and cancer. Oxidative damage to the mitochondrial membrane may also occur during the development of the disease, resulting in membrane depolarisation and the uncoupling of oxidative phosphorylation, which, in turn, alters cellular respiration . Whereas ROS and RNS are capable of targeting a variety of subcellular components, mitochondria are exposed rapidly and constantly to ROS and suffer oxidative damage to a greater extent than the rest of the cell. In fact, mitochondrial membranes, proteins and mtDNA appear to be particularly sensitive to oxidative and nitrosative damage .
ROS are reported to induce a multiple effects, including preferential and sustained mtDNA damage, altered mitochondrial transcript levels and mitochondrial protein synthesis [30-31]. ROS and RNS have been implicated in cancer, and increased oxidative stress is a common characteristic of many diseases and cancer risk factors, as they mediate post-translational modifications of mitochondrial proteins [31-32]. Exogenous and endogenous sources of NO are known to inhibit mitochondrial respiration (O2 consumption), thereby increasing O2-Â· production. At the same time, the "steepness" of the O2 gradient from the vascular lumen decreases , which mediates different transcription factors, such as hypoxia inducible factor (HIF-ï¡) .
Mitochondrial damage interferes with the capacity of the cell to generate energy, and with redox signalling and a variety of important functions regulated by mitochondrial ROS generation and response. ROS generated in the ETC have been highlighted as intermediate messengers of the activation of NF-ï«B [35-36]. This hypothesis is supported by the fact that cells lacking mitochondria also exhibit significant suppression of NF-ï«B activation. Furthermore, it is possible that mitochondrial damage modifies the effects of signalling factors. Hence, cellular response and function depend on the relative balance between the stimuli of mitochondrial ROS production and the concomitant accumulation of organelle damage, and thus the cellular response to increased oxidative stress can provoke disease. Consequently, mitochondrial damage would appear to constitute a general, yet direct, index or predictor of mitochondrial dysfunction.
Targets in the electron transport chain
The mitochondrion comprises a matrix surrounded by two membranes, the mitochondrial inner membrane (MIM) and the mitochondrial outer membrane (MOM). The MIM includes multiple invaginations and is highly impermeable to small molecules and ions, which require specific transport proteins to enter or exit the mitochondrial matrix. Under aerobic conditions, the proteins of the electron transport chain (ETC), located in the MIM, reduce oxygen to water through a series of steps along the electron transport chain that employ NADH and FADH2 derived from the tricarboxylic acid cycle and glycolysis. These reductions effectively pass protons (H+) across the MIM such that they accumulate in the inter-membrane space (IMS) creating a pH gradient across the MIM that contributes to an overall electrochemical gradient (DC). This gradient is used by the mitochondrial F1F0-ATPase as a source of energy to drive the synthesis of ATP from ADP and phosphate. This sequence of chemical steps is collectively known as oxidative phosphorylation. As a result of incomplete oxygen reduction, ROS are generated during normal oxidative phosphorylation. A high NADH:NAD+ ratio (as may arise owing to high rates of glycolysis) can increase ROS production, as does state 4 respiration in which electron transport occurs in the absence of ATP synthesis, for example, when ADP levels are low . Furthermore, inhibitors of the electron transport chain and of the F1F0-ATPase can also increase mitochondrial ROS production. ROS contribute to oxidative damage of cellular macromolecules, but in addition act as secondary messengers with important signaling roles . It is also noteworthy that the production of ROS has been identified as a common mechanism for the bactericidal effect of many widely used antibiotics including drugs targeting protein synthesis, DNA, and the cell wall . Thus, ROS perform both a necessary role and a destructive role in cells.
Inhibitors of the electron transport chain are useful tools for furthering our understanding of this essential bioenergetic process . Inhibitors of complex I (NADHubiquinone oxidoreductase) include rotenone, and the phytochemical Annonaceous acetogenins have been attributed with antimicrobial and anticancer properties. The widely used diabetes drug metformin inhibits complex I and has been shown to induce a p53- and AMP-activated protein kinase-dependent increase in glycolysis to compensate for modulation of the respiratory chain, which effectively increases glucose consumption . Complex II (succinate-ubiquinone oxidoreductase) is one proposed target of redox-silent vitamin E analogs such as ï¡-tocopheryl succinate . Complex III (cytochrome c oxidoreductase) is inhibited by antimycin A and by the natural product myxothiazole. Complex IV (cytochrome c oxidase) is a target of cyanide. Complex I and complex III are the major sources of mitochondria derived ROS in vitro, although the synthesis of superoxide by complex III is considered to be more physiologically relevant . The electron transport chain supplies the H+ gradient that is necessary for the mitochondrial F1F0-ATPase to function. Oligomycin, a natural product that blocks the proton channel, and the related macrolide apoptolidin, are both inhibitors of the F1F0-ATPase . Apoptolidins display remarkably selective cytotoxicity toward a subset of tumor cell lines in vitro, suggesting that inhibition of the ATPase is not indiscriminately cytotoxic. Other compounds reported to bind to the F1F0-ATPase include Bz-423 , resveratrol , diindolyl methane (DIM) , aurovertin , and PK11195 . The benzodiazepine derivative Bz-423 was identified as a lead for the treatment of autoimmune diseases . It reduces disease in murine models of lupus, arthritis, and psoriasis and has anti-proliferative and cytotoxic effects on tumor cells in vitro . Bz-423 is an uncompetitive inhibitor of the F1F0-ATPase, slowing the ATPase without causing a significant drop in cellular ATP levels . The therapeutic effects of this compound are mediated by the induction of superoxide. Resveratrol, a constituent of grape skins, increases longevity in rodents  and has been attributed with beneficial effects against cancer, heart disease, and inflammation . F1F0-ATPase inhibitors that specifically block ATP hydrolysis without affecting ATP synthesis have been described: such compounds should be effective under ischemic conditions when the ATPase can operate in the reverse of its normal direction leading to a catastrophic drop in ATP levels that causes cell death . Bacterial and mammalian ATP synthases exhibit substantial differences in structure and intracellular location presenting the opportunity for species selective ATP synthase modulation.
Targets in the mitochondrial permeability transition pore
The mitochondrial outer membrane is more permeable to small molecules than the the inner membrane. For this reason, the inter-membrane space resembles cytosol in its molecule composition. However, the inter-membrane space contains proteins such as cytochrome c, smac/Diablo (second mitochondria derived activator of caspases/direct inhibitor of apoptosis binding protein with low isoelectric point), and apoptosis inducing factor (AIF) that when released into the cytosol induce apoptosis by activation of caspases. One process for the release of these death inducing protein factors involves swelling of the mitochondrion so that the outer membrane ruptures producing mitochondrial outer membrane permeability (MOMP). These events are mediated by the mitochondrial permeability transition pore (MPTP), which comprises several proteins including the adenine nucleotide transporter (ANT) located in the MIM, a voltage-dependent anion channel (VDAC) located in the MOM, as well as the peripheral benzodiazepine receptor (PBR), hexokinase, cyclophilin D, and possibly also Bcl-2 and Bax . Inhibitors of the MPTP have been reviewed elsewhere as have inhibitors of bcl-family proteins . High affinity ligands of the PBR have been associated with anticancer and immunotherapeutic properties . VDAC ligands identified in cell-based screens have been shown to be selectively cytotoxic toward cells bearing oncogenic Ras protein . Redox cellular redox potential can be altered by proliferative state as well as by the function of OXPHOS proteins . Elevations in intracellular oxidant potential can have discrete chemical consequences: for example, a pair of cysteine thiols in the ANT becomes oxidized to a disulfide linkage that results in a conformational change and opening of the MPTP. Thus, manipulating cellular redox represents an approach to altering mitochondrial function. Elesclomol TM (STA-4783), an injectable drug undergoing Phase III clinical evaluation for the treatment of metastatic melanoma, selectively kills cancer cells through apoptosis as a result of an increase in their already raised oxidant level . Complementary to the use of pro-oxidant molecules is the application of inhibitors of proteins designed to maintain cellular redox by reducing ROS, for example, superoxide dismutase (SOD), catalase, and various peroxidases. 2-Methoxyestradiol magnifies the effects of cytotoxic agents and displays anti-leukemic activity as a single agent in culture stemming from accumulation of ROS, which is proposed to be due to its inhibition of SOD . By contrast, persistent mitochondrial production of ROS leading to persistently increased oxidant stress and mitochondrial damage has been linked to many differenet diseases such as degenerative diseases and aging . This free radical theory of aging suggests that a reduction in ROS should have therapeutic benefit. Indeed insulin sensitivity and glucose homoeostasis was improved in obese insulin-resistant mice treated with the antioxidants N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) .
A wide range of antioxidants could be targeted to mitochondria by conjugation to the triphenylphosphonium (TPP) moiety. These include TPP-conjugated derivatives of ubiquinone , tocopherol , lipoic acid spin traps and ebselen . Research has tended to focus on antioxidants that are effective against lipid peroxidation, because this feature is very important in mitochondrial oxidative damage and alkylTPP conjugates are closely associated with the mitochondrial inner membrane. In particular, the the most extensively evaluated and best understood member of the family targeted version of ubiquinol is (MitoQ) . MitoQ is taken up rapidly by isolated mitochondria driven by the mitochondrial membrane potential (Î”Ïˆm), and, within mitochondria, nearly all of the accumulated MitoQ is adsorbed to the matrix surface of the inner membrane. MitoQ is reduced to the active ubiquinol antioxidant by complex II of the respiratory chain. It cannot restore respiration in mitochondria lacking coenzyme Q because the reduced form of MitoQ is not oxidized by complex III. Consequently, all of the effects of MitoQ are probably attributable to the accumulation of the antioxidant ubiquinol form. Importantly, when the ubiquinol form of MitoQ acts as an antioxidant, it is oxidized to the ubiquinone form. This ubiquinone form is then rapidly re-reduced by complex II, which restores its antioxidant efficacy. As MitoQ is generally adsorbed to the mitochondrial inner membrane, and given that its linker chain enables its active ubiquinol antioxidant component to penetrate deeply into the membrane core, it is thought to be an effective antioxidant against lipid peroxidation. MitoQ has also been shown to protect against ONOO- damage . In addition, it can react with O2-, however, as in the case of other ubiquinols, its reactivity with H2O2 is negligible. In summary, in isolated mitochondria, MitoQ seems to fulfil most of the requirements of a mitochondria-targeted antioxidant .
Mitochondria-targeted antioxidants have been used in isolated cells and importantly, toxicity is the first aspect to be considered when moving from isolated mitochondria to cells. The extensive accumulation of lipophilic cations within isolated mitochondria can disrupt membrane integrity, respiration and adenosine 5'-triphosphate synthesis as a consequence of the adsorption of the cations to the matrix surface of the inner membrane. Supporting this idea, the more hydrophobic TPP cations disrupt mitochondrial function at lower concentrations, and the degree of this effect correlates with the amount of compound adsorbed to the inner membrane. The non-specific effects of MitoQ on mitochondria are assessed using the control compound decylTPP, which is similar to MitoQ in hydrophobicity but lacks the antioxidant ubiquinol moiety. The non-specific disruption of the mitochondrial function induced by MitoQ and decylTPP occurs at similar concentrations and limits the amounts of TPP-derived targeted antioxidants that can be used. Therefore, it is imperative that these compounds exert their antioxidant effect at concentrations well below those that disrupt mitochondrial function. When comparing cells to isolated mitochondria, it is important to bear in mind that, due to the plasma membrane potential, targeted antioxidants are accumulated within cells at a concentration 5- to 10-fold greater than that of the extracellular environment . For mammalian cells in culture, this concentration is generally in the range of 0.1-0.5 Î¼M, but can vary considerably with cell density, cell type, and incubation conditions. MitoQ uptake into cells is largely blocked if the Î”Ïˆm is inhibited with the uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). This is consistent with the fact that the cellular uptake of MitoQ is primarily driven by the mitochondrial distribution of the drug rather than its accumulation in other cell compartments. A further indication of the membrane potential-dependent mitochondrial concentration of TPP-containing molecules within cells is provided in a cell model of Friedreich's ataxia, in which the protection afforded by MitoQ was shown to be FCCP-sensitive whereas the same uncoupler did not affect the potency of the non-targeted compounds decylQ or idebenone . Therefore, upon incubation with cells in culture, MitoQ is accumulated predominantly within the mitochondria, but the amount of MitoQ present in the cell is currently difficult to quantify. MitoQ has been used in a large range of mitochondrial and cell models  and has been shown to provide protection against oxidative damage. The interaction of MitoQ with mitochondrial ROS within rotenone-treated fibroblasts has been studied in detail . MitoQ did not decrease O2- production when measured by dihydroethidium oxidation, but it did prevent lipid peroxidation when measured by the fluorescent probe C11-BODIPY . This finding is consistent with the model for MitoQ action based on studies with isolated mitochondria which have demonstrated that MitoQ's main antioxidant action is to prevent lipid peroxidation. Nevertheless, it remains to be seen whether this is the primary mechanism by which MitoQ acts as a protective agent in all cell types and all forms of oxidative stress.
Mitochondria-targeted antioxidants have also been used in in vivo studies. To function as therapy, mitochondria-targeted antioxidants must be delivered to mitochondria within the cells of patients, preferably following oral administration. As TPP cations pass easily through phospholipid bilayers, they pass from the gut to the bloodstream and from there to most tissues. It has been shown that, when simple alkylTPP compounds such as the targeted version of Vitamin E (MitoE) or MitoQ10 are administered intravenously to mice, they are rapidly cleared from the plasma and accumulate in the heart, brain, skeletal muscle, liver and kidney . Importantly, TPP-derived compounds have been shown to be orally bioavailable as feeding mice by adding tritiated methylTPP, MitoE2 or MitoQ in their drinking water, led to uptake into the plasma, and from there into the heart, brain, liver, kidney and muscle . To summarize, it is possible to administer alkyl-TPP compounds to animals orally, as these compounds are taken up into the plasma with reasonable bioavailability and are then rapidly cleared from the plasma when they accumulate in mitochondria within the tissues. Having shown that the long-term administration of mitochondria-targeted antioxidants is viable, the next step was to determine whether the amount of compound accumulated is sufficient to act as an antioxidant in vivo. Thus, in another study, rats were administered 500 Î¼M MitoQ for 2 weeks via their drinking water. Their hearts were then isolated and exposed to ischemia-reperfusion injury in a Langendorff perfusion system, and MitoQ was shown to provide greater protection against the loss of heart function, tissue damage and mitochondrial function than methylTPP or short-chain quinol controls . The most probable reason for the protection observed in these experiments is that lipid peroxidation in the mitochondrial inner membrane was prevented by MitoQ . However, this has yet to be confirmed by showing that MitoQ blocks any increase in markers of oxidative damage in mitochondria. Taken as a whole, this evidence suggests that mitochondria-targeted antioxidants are effective in reducing oxidative stress in atherosclerosis and could attenuate lesion/plaque development.
The development of MitoQ as a pharmacological agent is proving to be somewhat different from that of most other drugs. Typically in medicinal chemistry, research focuses on lead compounds that interact with a specific target, such as a receptor binding site. In assessing these compounds, a preliminary screening is often carried out to ensure that drug candidates are soluble, bioavailable and can pass through phospholipid bilayers . However, mitochondria-targeted antioxidants based on TPP lipophilic cations are less constrained by these traditional guidelines, as they have the unusual property of being both relatively water soluble and membrane permeant. Even though the molar mass of MitoQ is relatively large for a pharmaceutical agent, and despite its high octanol-PBS partition coefficient, it is readily bioavailable and passes easily through biological membranes. A further unusual feature of TPP-targeted compounds is that they are targeted to an organelle to modify a general, rather than specific, process (namely oxidative damage). Therefore, if lipophilic cations such as MitoQ prove to be effective drugs, a new approach to medicinal chemistry and drug discovery would be established. MitoQ is being developed as a pharmaceutical agent by Antipodean Pharmaceuticals Inc. (http://www.antipodeanpharma.com/). In the search for a commercially satisfactory stable formulation, a compound was created with the methane sulfonate counter anion and decomposition was inhibited by complexation with Î²-cyclodextrin. This preparation is easily manufactured as tablets, and has passed conventional animal toxicity tests with no observable adverse effects at 10.6 mg/kg. In Phase 1 trials, MitoQ has shown a positive pharmacokinetic behaviour when administered orally at 1 mg/kg, which results in a plasma Cmax = 33.15 ng/mL and Tmax at approx. 1 h. This formulation has good pharmaceutical characteristics and is now in Phase 2 trials for Parkinson's disease and hepatitis C .
Mitochondrial selectivity and toxicity
Given the importance of mitochondria to cellular bioenergetics and homeostasis, it should not be surprising that shutting down mitochondrial respiration can be detrimental . In addition, since mitochondria possess their own DNA, compounds that target DNA can also perturb mitochondrial integrity, including intercalators (e.g. ethidium bromide), reverse transcriptase inhibitors (e.g. AZT) and topoisomerase inhibitors (e.g. ciprofloxacin and etoposide). Non-nucleoside reverse transcriptase inhibitors, used in the treatment of HIV-AIDS, affect mitochondrial function by inhibiting DNA pol g, which is required for the replication of mtDNA . The lactic acidosis and idiosynchratic hepatic failure associated with these drugs has been attributed to their effects on mtDNA. Delocalized lipophilic cations such as F16 andMKT-077 can dissipate the mitochondrial membrane gradient by facilitating anion flux across the MIM, inhibiting ATP synthesis. A purported association between mitochondrial toxicity and either withdrawal from the market or a black-box label, has prompted calls to screen for mitochondrial toxicity early in the drug discovery process in order to reduce late-stage attrition . Notwithstanding these concerns, the electron transport chain has evolved to allow for some suppression in the activity of individual complexes without a net reduction in the overall rate of respiration . Moreover, neoplastic cells and immune receptor-activated lymphocytes undergo a metabolic switch to increased glycolysis and may be less sensitive to the inhibition of OXPHOS . At the same time, cells in which electron transport and ATP synthesis are weakly coupled or that otherwise generate higher than normal oxidant levels, are more sensitive to a pharmacological increase in ROS and more prone to apoptosis . Thus, the production of ROS can have profoundly different consequences depending on context.
DIABETES AND OXIDATIVE STRESS
Diabetes mellitus (DM) is a growing health problem worldwide. It is associated with different complications that affect the quality of life and life expectancy of patients. Today, 250 million people around the world live with diabetes, and this figure is expected to rise to 400 million over coming years. Logically, if diabetes is related with an increase in the risk of cancer, the potential clinical consequences for the population are serious. Cancer and diabetes are related and it has been investigated widely, and the mayority of evidence suggests that DM is associated with risk of different types of cancer. However, the majority of the studies require careful reinterpretation, as DM is not a single disease, but can be any one of a group of different metabolic disorders characterized by an increase of glucose levels. Taking into account this general context, each type of diabetes has its individual metabolic and hormonal abnormalities that can affect each patient in different ways. It is therefore risky to evaluate diabetic patients as a homogeneous cohort. Furthermore, a series of parameters potentially related to this condition (population, statistically methods, obesity, quality of metabolic control, drugs employed for treatment, diet, etc.) may have a bearing on the association between diabetes and cancer. However, it can be said that oxidative stress is generally linked to diabetes, and diabetes is characterised by mitochondrial impairment [10-11].
Cancer risk and diabetic patients
A series of studies and meta-analyses confirm an elevated risk of diseases (including liver, pancreas, colorectal, kidney, bladder, endometrial and breast cancers, and non-Hodgkin's lymphoma) in diabetic patients (Table 1). A reduced incidence of prostate cancer has been reported in diabetic patients (Table 1), but there is no evidence of an association with other cancers. Diabetes is an underdiagnosed disease (around 5% of the adult population has undiagnosed diabetes; , and so any control population is likely to include such individuals, which will increase the risk of cancer among the 'normal' population. In diabetic patients, cancer may be favoured by: i) general mechanisms that promote the onset or progression of cancer in any organ due to associated alterations (i.e. hyperglycemia or hyperinsulinemia or drugs) that affect all tissues; and ii) site-specific mechanisms affecting cancerogenesis of a particular organ.
Inflammation and oxidative stress
Diabetes is related to inflammation and oxidative stress, and its implicates a high production of pro-inflammatory cytokines such as TNFï¡ and IL-6. This chronic pro-inflammatory state (which persists for long periods of time) reduces intracellular anti-oxidant capacity, leaving susceptible cells vulnerable to malignant processes. In fact, high concentrations of diverse ROS can damage different molecules such as cell DNA by direct oxidation or by interfering with the mechanisms of DNA repair . ROS may also damage proteins and lipids, forming derivative subproducts that alter homeostasis, thus favouring the accumulation of mutations that, in turn, contribute to the multistage carcinogenesis process . It is possible the appearance of other mechanism which is related to mitochondrial dysfunction, a well-recognized abnormality in diabetes. DNA repair is a high energy-consuming process that requires increased mitochondrial activity: stimulating malfunctioning mitochondria not only results in a low, insufficient energy supply, but also increases ROS production . Moreover, an additional factor correlated with insulin resistance is the pro-inflammatory cytokine TNFï¡ produced by adipose tissue . TNFï¡ induces development and progression of many tumours  by the action on different transcription factors such as nuclear factor-kappa B (NF-ï«B), which it is involved in pro-tumoral effects of TNFï¡. In conclusion, through mechanisms specific to both diabetes and other chronic degenerative diseases, DM may accelerate the aging biological processes that promote cancerogenesis.
Mitochondrial respiratory chain uses glucose via oxidative phosphorylation. Glucose is converted to differentes substartes such as pyruvate. During this process, NADH and FADH2 are transported into the mitochondria via different shuttle systems. With high amounts of glucose which means hyperglycemia, there is an increase in the NADH/NAD+ with deletereal complications. In fact, NADH is one of the main electron donor to the mitochondrial electron transport chain . Therefore, are necessary therapies that would decrease the hyperglicemia in cells in order to have benefit in diabetes complications  by decreasing the substrates fuel availability to mitochondria. Mitochondria also utilize another substrates such as free fatty acids (FFAs) as fuel, and their oxidation in the tricarboxylic acid cycle generate NADH and FADH2; so, the high levels of FFAs can mimick the mitochondrial impairment induced by hyperglicemia.
It is known, that in established renal disease, control of cholesterol, triglycerides and dietary fat intake with HMG-CoA reductase inhibitors should defend the mitochondria from oxidative stress. The generation of ROS is recognized as one of the main factors implicated in the development of diabetes complications  being the mitochondria de main source. Therefore, the use of molecules to erradicate these mitochondrial ROS generation has increasingly been considered as a relevant aim in ameliorating the complications of diabetic disease. During oxidative phosphorylation, almost 90% of oxygen is metabolized, and electrons from different substrates are transferred to molecular oxygen, involving electron transport chain including complexes I-Ivcomplexes and ATP synthase. During the oxidative phosphorylation, protons are pumped across the mitochondrial membrane and voltage gradient is generated in order to generate ATP. There aretwo main sites of electron leakage which are complex I and III of the electron transport chain . For this reason, in diabetes, where there is high amount of substrates supplied as a consequence of high levels of glycemia it has been speculated , that excess production of ROS has an important effect in the mitochondrial membrane potential which, rather than ATP synthesis, leaking electrons to form ROS. While these findings are very important, these predominantly tissue studies  remain to be fully supported in vivo. Specifically, dysfunction and complications of the mitochondrial respiratory chain has been hypothetized to contribute to many different pathologies, and patients with deleterous genetic mutations that implicate a decrease in the activity of complex I, have highly rates of mitochondrial ROS production . In this sens, it has been described that patients with diabetes develop mitochondrial dysfunction in complex I, and as a consequence there is an increase in ROS production, and a decrease in the antioxidant levels and membrane potential . In other pathologies which develop insulin resistance such polycystic ovary syndrome, it has been also described that there is a n impairment in mitochondrial complex I .
There are more evidences about the role of mitochondrial oxidative phosphorylation as a one of the main candidates in the development of diabetes complications such as the disease Friedreich's ataxia, which is a genetic disorder due to mutations causing a down regulation of mitochondrial complex I and a highly mitochondrial ROS generation . In another study, it has been described that around 50% of children with mitochondrial impairment have renal diseases suggesting that in the diabetic kidney disease, the mitochondria play a very important role . In addition, it has been demonstrated that in patients with several defects in the electron transport chain, the renal disease is their primary pathology . This results suggest that future research investigation in mitochondrial impairment is warranted and is of research priority. In this sense the therapy with mitochondria-targeted antioxidants would be beneficial. The mitochondrial ROS production initiates a range of damaging reactions which can then damage different molecules or structures such as lipids, proteins, and nucleic acids. In fact, in the mitochondria there are many enzymes susceptible to damage by ROS inducing impairment in the cellular calcium storage, diminshed ATO production and changes in the membrane potential, all of them are involved in the development of apoptosis or necrosis. For example, idebenone is a new mitochondrial antioxidant that has a high availability into organs. This kind of antioxidant is a safe and very efficient way to protect mitochondrial function from oxidative damage in humans with Friedreich's ataxia  suggesting their potential as a therapeutic tool. Interestingly, idebenone also reduces cardiomyopathy in these subjects unlike traditional antioxidants such as alpha-tocopherol or vitamin E .
MitoQ is other mitochondria-targeted antioxidant with selective uptake into mitochondria due to its covalent attachment of its antioxidant moiety to the lipophilic triphenylphosphonium cation. This molecule accumulates in mitochondria, and the changes in the membrane potential can increase its uptake by 500-fold . The efficacy of these targeted-mitochondria antioxidants in diabetes remains to be determined; however, their targeted specificity for mitochondria suggests that this is an interesting research topic for the treatment of diabetes and cardiovascular diseases.
In this sense, it has recently published that orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period improves tubular and glomerular function in the Ins2(+/)â»(AkitaJ) mice (an animal model of diabetes). MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Furthermore, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and Î²-catenin showed a nuclear accumulation in the Ins2(+/)â»(AkitaJ) mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy .
Uncoupling of the respiratory chain.
The collapse of the mitochondrial membrane potential is a very important parameter to determine the possible appearance of apoptosis or necrosis, and this effect can occur via uncoupling of the electron transport chain where energy is used to generate heat rather than for ATP synthesis. Indeed, chronic uncoupling decreases ATP synthesis and increases the leakage of electrons to oxygen to form ROS and therefore oxidative stress. Uncoupling proteins consist of three major isoforms, UCP-1 to -3, that bind to the electron transport chain at the location of ATP synthase. It has been suggested in different studies involving diabetic neural tissues or retinal endothelial cells that overexpression of uncoupling proteins is responsible for the "back up" of electrons in the respiratory chain and their leakage to ROS. Therefore, therapeutic agents which can that decrease the amounts of these uncoupling proteins, thereby lowering mitochondrial ROS generation, may lead to a novel therapy for renal and neural diseases implicated in diabetes. In this line, this therapy has been used successfully in experimental models of disease . Therefore, the challenge is to have an agent that modulates mitochondrial ROS production without compromising ATP generation and maintaining the cell viability.
Organisms have developed numerous antioxidant systems including ROS and enzymes, in response to excess ROS production and oxidativ stress during respiration and metabolism (Fig.). One of the most important of these antioxidant enzymes is superoxide dismutase (SOD). There are three major cellular forms: copper zinc (CuZnSOD, SOD1), manganese (MnSOD, SOD2), and extracellular (SOD3). These enzymes are responsible for the detoxification of superoxide to H2O2 and water. Catalasa and glutathione peroxidase (GPx) are other antioxidant enzymes that catalyze the conversion of H2O2 to water. There are also numerous other antioxidants present in cells, such as glutathione and different vitamins which have important function in the homeostasis of the cells. Furthermore, a number of these antioxidants have proven to play a poor neneficial effects in the treatment of diabetic complications. In fact, it has been described that in diabetic microvascular disease, there is a decrease in the activity and expression of each of these antioxidant enzymes . For example, the overexpression of CuZnSOD showed beneficial effects against organ impairment in models of type 2 diabetic nephropathy . In other studies in animals with genetic deletions of antioxidant enzymes have also showed low beneficial effect of MnSOD  to the development of diabetes complications. In one study, it has been described that MnTBAP (MnSOD mimetic) has also shown efficacy in preventing ROS-induced injury in vitro , although the use in vivo of such drugs has shown confusing data  because of lacking of substantial beneficial effects. It is important to point out that specific polymorphisms of the MnSOD gene are correlated with the development of diabetic nephropathy . Interestingly, in an animal model of GPx-1-deficient mice, it has been described that there is no increased risk for microvascular disease, and in particular diabetic nephropathy [104. Overexpression of other antioxidant enzymes such as catalase in experimental models of type 2 diabetic nephropathy looks to be protective  guggesting the importance to erradicate the elevated levels of H2O2. However, in humans studies, it have been indicated no relationship between catalase gene polymorphisms and the incidence of nephropathy in diabetes (Fig. 2) .
ROS PRODUCTION AND DIABETIC CPMPLICATIONS
There are many evidences which suggest that oxidative stress play a key role in the development and progression of diabetic complications such as nephropathy (Fig.). The use of different agents in order to modulate ROS generation would include a decrease in the cellular uptake of glucose and afterwards retarding the feeding of glucose metabolites in the oxidative phosphorylation. Having said that, however, it is considered that oxidative phosphorylation and mitochondrial function are key targets to modulate the progression of diabetic complications such as nephropathy. The high levels of glucose can induce mitochondrial ROS, which lead to activation of different biochemical pathways, hexosamine, increased flux through the polyol, increased AGE formation and activation of protein kinase C isoforms . However, there are another important pathways such as NADPH oxidase or the uncoupling of eNOS which need further investigation to study their relative importance in progressive diabetic complications such as renal disease.
We thank B Normanly for his editorial assistance. This study was financed by grant PI10/1195, PI09/01025, CIBERehd. VMV and MR are the recipients of Fondo de Investigacion Sanitaria (FIS) contracts (CP07/00171 and CA07/00366 respectively).