Re:Mitochondrial Dysfunction and Redox Imbalance as a Diagnostic Marker of “Free Radical Diseases”

[문형철]

beyond reason

활성산소질환에 의한 미토콘드리아 문제의 진단

Mitochondrial Dysfunction and Redox Imbalance as a Diagnostic Marker of “Free Radical Diseases”

1. EKATERINA GEORGIEVA¹, 
2. DONIKA IVANOVA¹, 
3. ZHIVKO ZHELEV¹,², 
4. RUMIANA BAKALOVA³,⁴,⁵⇑, 
5. MAYA GULUBOVA¹ and 
6. ICHIO AOKI³

+Author Affiliations

1. ¹Medical Faculty, Trakia University, Stara Zagora, Bulgaria
2. ²Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria
3. ³Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences (NIRS), QST, Chiba, Japan
4. ⁴Group of Quantum-state Controlled MRI, National Institute of Radiological Sciences (NIRS), QST, Chiba, Japan
5. ⁵Medical Faculty, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria

1. Correspondence to: Dr. Rumiana Bakalova, Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences (NIRS), QST, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Tel: +81 8034307755, e-mail: bakalova.rumiana@qst.go.jp

 

Next Section

Abstract

The intracellular redox balance (redox status) is a dynamic system that may change via many factors. Mitochondria are one of the most important among them. These organelles are the main intracellular source of energy. They are essential for maintaining cellular homeostasis due to regulation of many biochemical processes. The mitochondrial dynamics change during cellular activities and in some cases, can cause an overproduction of reactive oxygen species (ROS), which encourages the induction of oxidative DNA damage and up- or down-regulation of phosphatases, proliferative/anti-proliferative factors, apoptotic/anti-apoptotic factors, etc. 

Moreover, mitochondrial dysfunction and redox imbalance can continuously support and contribute to a wide range of pathologies, termed as “free radical diseases” (e.g., cancer, neurodegeneration, atherosclerosis, inflammation, etc.). This review article is focused on the mitochondrial dysfunction and cellular redox status as a hallmark of cell homeostasis and diagnostic marker of cancer. It is intended to broad readership – from students to specialists in the field.

• Mitochondrial dysfunction
 
• redox status
 
• free radical diseases
 
• review

Previous SectionNext Section

Reactive Oxygen Species, Redox Imbalance and Oxidative Stress

Maintaining the level of reactive oxygen species (ROS) in a balanced state and proper functioning of redox systems are crucial for redox status in the living cells. In the organism, the redox balance is based on the generation and elimination of ROS by endogenous and exogenous sources (1). Normal cells of healthy mammals are characterized by low steady-state levels of ROS and constant levels of intracellular reducing equivalents (2). ROS are universal products of aerobic metabolism, which can be generated during cellular respiration or as a result of specific enzymes that seem to be centrally involved in redox signalling (3, 4). The balance between generation of ROS and their neutralization by endogenous cellular defense mechanisms is crucial for maintaining normal cell homeostasis, because some types of ROS (e.g., superoxide, hydrogen peroxide, nitric oxide) serve as signaling molecules (5, 6). Low/moderate levels of ROS are involved in normal biochemical pathways: (i) cellular response against infections; (ii) intercellular recognition and signal transduction; and (iii) induction of mitogenic response (7-9). Abnormal generation of ROS induces oxidative stress and “free radical pathologies” via damages of biological macromolecules and genotoxicity (10). The carcinogenesis is a classical example. As a result of intracellular redox imbalance, the abnormal levels of ROS may lead to cell dysfunction – inhibition of protein phosphatases and activation of protein kinases, malignant transformation and tumor development and progression (11, 12) (Figure 1). These events are accompanied by activation of transcription and translation factors, accumulation of defective proteins, and adaptation to high levels of ROS and resistance to ROS-dependent apoptosis – a common behaviour of all cancer cells (13). Many authors indicate that the oxidative stress is a direct or indirect result of ROS-mediated damage on biological macromolecules and a major factor not only for carcinogenesis, but also for neurodegeneration, cardiovascular disease, inflammation, atherosclerosis, diabetes, aging, etc. (14-20).

The disruption of cellular homeostasis by activation of oncogenes, mitochondrial dysfunction and/or oxidative stress leads to an increased genomic instability and the target cells are able to adapt to the changing environment (21). As a result, we observe several events: enhancement of cell proliferation, angiogenesis, and metastatic potential, which induces cancer development (22). In turn, chronic inflammatory processes, caused by biological, chemical, and physical factors, are also associated with an increased risk of developing a variety of malignancies (Figure 2) (23).

Historical origins of the concept of redox status used to determine the ratio between the mutually convertible oxidized and reduced forms of a specific endogenous redox-pairs relative by the Nernst equation (24). In addition to determination of the redox potential of different redox-pairs, the Nernst equation can be an instrument for providing a quantitative assessment of various intracellular redox systems and to eval‎uate the cellular redox status (25). At a later stage, the “redox status” as a term in redox biology and medicine is widely used to describe oxidation-reduction changes, caused by free radicals and oxidative stress.

Cancer and non-cancer cells are characterized by an entirely different redox status, which is the basis of diagnostics and development of new therapeutic strategies. It is widely accepted that a moderate increase in ROS can promote cell proliferation and differentiation, but extremely excessive amounts of ROS can cause irreversible oxidative damages of biomacromolecules, apoptosis and cell death (26). Therefore, maintaining ROS homeostasis at low levels is crucial for normal cell survival, while the moderate enhancement of ROS is associated with adaptation, abnormal cancer cell growth and conservation of redox imbalance. Prolonged operation of cells at abnormal steady-state levels of ROS provokes genetic mutations, which makes them well-adapted to oxidative stress. This process is in the basis of malignant transformation. The cancer cells develop an enhanced endogenous antioxidant capacity. The cells that survive intrinsic oxidative stress mobilize a set of adaptive mechanisms, which not only activate ROS-scavenging systems to fight with the oxidative stress, but also to inhibit apoptosis. Recent evidences suggest that such adaptation contributes to malignant transformation, metastasis and resistance to anticancer drugs (26-30).

Which is the main endogenous source of ROS and how the substantial biochemical difference between normal and cancer cells and tissues could be used as a diagnostic marker and therapeutic target?

Previous SectionNext Section

Role of Mitochondria in “Free Radical Pathologies”

Mitochondria play a central role in the regulation of cellular bioenergetics, which is responsive to changes in the environment, caused by hormones, nutrients, partial oxygen pressure, oxygen amendments and others (31, 32). They are essential for cell viability. However, mitochondrial gene mutations, which are often found in cancer cells, can impair mitochondrial energy metabolism or mitochondrial bioenergetics and biosynthesis, change the status and serve as a trigger of mitochondrial “retrograde signaling” of the nucleus (33, 34). The mitochondrial redox control is important not only for oxidative phosphorylation, ATP synthesis, calcium homeostasis, thermogenesis, apoptosis and ROS production, but it affects the redox balance of the entire cell (35). Mitochondria not only provide energy for the cell, but also participate in many other cellular functions, including calcium signalling, membrane potential regulation, heme and steroid synthesis, cell proliferation, apoptosis, etc. (36). Thus, mitochondria are the main source of intracellular ROS (e.g., superoxide and hydrogen peroxide) (Figure 3) (26, 37-41). The production of ROS in mitochondria is tightly regulated by the mitochondrial superoxide dismutase (SOD2) and glutathione-peroxidase (GPx), as well as by catalase and non-enzymatic antioxidants.

Overproduction of ROS in mitochondria and alteration of mitochondrial dynamics can promote carcinogenesis through suppression of complex I, induction of oxidative mitochondrial DNA damage, increased excessive calcium ion influx, inhibition of key phosphatases, induction of key kinases and transcription factors (42, 43). Data indicate also a key role of mitochondrial dysfunction in cardiovascular pathology and aging. Mitochondrial damage could trigger ROS overproduction, leading to detrimental structural and functional effects on the cardiovascular system (44). An increasing number of studies have demonstrated that mitochondrial oxidation of thiol-containing proteins is a major event in myocardial infarction and stroke. A basic characteristic of heart diseases, accompanied by oxidative stress, is mitochondrial dysfunction due to formation of ROS during reperfusion (45). Similar picture has been observed in aging, diabetes, atherosclerosis and neurodegeneration (46-49).

Substantial efforts were made in the understanding of the role of mitochondrial dysfunction and oxidative stress in these “free radical diseases”. Different theories of aging have been proposed by many researchers, including free-radical and mitochondrial theories of aging. Currently, one of the most widely-accepted explanations for the cause of aging is the gradual accumulation of dysfunctional mitochondria and oxidative damage with age (29). This mechanism is also one of the most widely discussed in carcinogenesis.

View larger version:

• In this page
 
• In a new window

Figure 1.

ROS-dependent malignant transformation in cells – a potential mechanism.

Previous SectionNext Section

Models of Oxidative Stress and Mitochondrial Dysfunction in Living Cells

Many studies suggest that superoxide has been implicated in the pathogenesis, while hydro-gen peroxide has been implicated in the apoptosis (50). Because the mitochondrial ROS are essential for both processes, cell signaling regulation and ROS-induced damage, we need a more detailed understanding and developing of methods for detection of redox dynamics in living organisms.

The most popular approach is to use a specific selective mitochondrial inhibitor to block electron leakage, especially from complex-I or complex-III of the electron-transport chain, e.g., antimycin A, cyanide, rotenone, myxothiazol, and oligomycin (51-59). Many researchers have used such experimental models of mitochondrial dysfunction to enhance drug-induced apoptosis in isolated mitochondria or intact cells. These studies were conducted to investigate the mitochondrial pathology and related oxidative damages (55, 60, 61). Mitochondrial in-hibitors of complex-I and complex-III cause a rapid increase in intracellular Ca²⁺, disruption of mitochondrial potential and depolarization of the mitochondrial membrane (59). For ex-ample, cyanide as an inhibitor of complex IV binds to the cytochrome c oxidase heme a3-CuB binuclear center to inhibit oxygen utilization in cells and compromises the oxidative phosphorylation and ATP synthesis (62-64). As a result of cytochrome c oxidase inhibition, a cascade of reactions is initiated, which leads to mitochondrial electron transport inhibition and overproduction of ROS at complexes I and III (65). Rotenone, cyanide, myxothiazol and oligomycin significantly inhibit resting background K⁺ by simulating the effects of hypoxia, in which leads to membrane depolarization (59).

In 2003, Pelicano et al. have described a basic strategy for exogenous induction of mitochondrial dysfunction in living cells, accompanied by overproduction of superoxide (Figure 4) (55). The authors have used simultaneously two specific compounds – rotenone and 2-methoxyestradiol (2-ME). Rotenone is an inhibitor of the electron flow through complex I and causes generation of superoxide. 2-ME is an inhibitor of mitochondrial SOD (Mn-SOD) and causes a further accumulation of superoxide. The combination of both leads to abnormal generation of superoxide radicals in the cells (55).

View larger version:

• In this page
 
• In a new window

Figure 2.

Role of low and high levels of ROS on cell response – survival or cell death.

View larger version:

• In this page
 
• In a new window

Figure 3.

Generation of ROS in the mitochondria (according to refs. 26 and 41). SOD: Superoxide dismutase; GPx: glutathione peroxidase; CoQ: coenzyme Q; Cyt c: cytochrome c; VDAC: voltage-dependent anion channel.

View larger version:

• In this page
 
• In a new window

Figure 4.

Experimental model for induction of mitochondrial dysfunction, overproduction of superoxide and oxidative stress by treating cells with rotenone and 2-methoxyestradiol.

View larger version:

• In this page
 
• In a new window

Figure 5.

Schematic presentation of the redox transformations of nitroxide radical in bio-logical systems.

Rotenone is a naturally-derived plant compound (66). Its main effect is to increase the pro-duction of mitochondrial ROS and to decrease the mitochondrial membrane potential and cellular ATP levels (67, 68). It can prevent a transfer of electrons from the Fe-S center of the mitochondrial NADH-dehydrogenase (complex I) to ubiquinone. As a result, rotenone de-creases overall ATP production and at the same time leads to production of abnormal levels of superoxide. The low levels of ATP and increased superoxide cause oxidative stress in the cells and consequently result in cell death (69). A number of commentaries have shown that rotenone is able to induce apoptosis via abnormal mitochondrial ROS generation in a variety of cells. The rotenone-dependent mitochondrial ROS induce apoptosis by DNA fragmenta-tion, cytochrome c release, and caspase 3 activation (70). The inhibition of neuronal mito-chondrial complex I with rotenone leads to an enhanced extracellular production of superox-ide and was primarily mediated by microglial NADPH-dependent oxidases in mice (71).

2-ME is a natural endogenous metabolite of 17β-estradiol that exerts anti-proliferative, anti-angiogenic, pro-apoptotic and transcriptional activity in various cells, including induction of cell-cycle arrest (72-76). Experiments have demonstrated that 2-ME is involved in the inhibi-tion of the polymerization of tubulin in vitro, thus disrupting normal microtubule function (77). Also, 2-ME leads to inhibition of mitosis at the metaphase and as a consequence – to the inhibition of cell proliferation and induction of apoptosis (78). 2-ME has been reported to have unique properties including cytotoxic, anti-proliferative and apoptotic effects in a vari-ety of malignancies (79). 2-ME can also stimulate pro-apoptotic factors and production of intracellular ROS. Another mechanism of action involves the inhibition of hypoxia-inducible factor (HIF) and interference with mitochondrial function by related compounds, which are inhibitors of complex I of the mitochondrial electron-transport chain (77). Specifically, 2-ME has been reported to inhibit the Mn-SOD, resulting in the release of cytochrome c from mitochondria and finally, in stimulation of caspases and generation of abnormal superoxide production (55, 80, 81). Thus, the treatment of cells with combination of rotenone and 2-ME is one of the best models for induction of mitochondrial dysfunction and oxidative stress in living cells.

Previous SectionNext Section

Nitroxides as Redox Sensors for Detection of Mitochondrial Dysfunction and Oxidative Stress in Cells and Tissues

The cyclic nitroxides, also known as aminoxyls, are stable free radicals that have unique chemical and biochemical properties. They are widely used as probes for measurement of oxygen, glutathione, pH change and redox status in living cells and tissues (82, 83). Nitroxide radicals are organic compounds containing an aminoxyl group (N-O^•) and have been used for many years as biophysical tools, due to the ability to interact with free radicals (Figure 5). They are stabilized by methyl-groups at the α-position in five-membered pyrrolidine and six-membered piperidine ring structures. Methyl-groups could be substituted with other groups (-R) on the ring. This produces a diverse range of compounds allowing modulation of specific properties (e.g., hydrophobicity, intracellular delivery, delivery across blood-brain barrier, etc.) and stability to reduction. For example, TEMPO derivatives with different substitutes at 4-position in the ring could influence the ROS scavenging activity and time for interaction with superoxide (84).

Many researchers have reported that stable nitroxide radicals are appropriate redox sensors for ROS imaging and undergo bioreduction to hydroxylamine derivatives, which can react with superoxide (85-87). For example, nitroxides as mito-TEMPO and mito-carboxy-PROXYL are mitochondria-targeted derivatives of TEMPOL and carboxy-PROXYL, which currently are widely used as redox-sensors for detection of abnormal superoxide generation in living cells and tissues (2, 28, 37, 88-91).

During the past 15-20 years, most studies have focused towards the biochemical interactions, biologically-relevant effects, and diagnostic and therapeutic applications of nitroxides. These studies generally describe their ability to degrade superoxide and hydroperoxides, to inhibit Fenton reactions and to undergo radical-radical recombination. Furthermore, they can alter the tissue redox status and to change the metabolic processes (92).

In vitro studies have demonstrated that various reducers or oxidizers may convert the contrast form of nitroxide radical to non-contrast hydroxylamine, which depends on physiological conditions (2, 28, 91, 93). Figure 5 shows the transformations between the oxidation radical state of nitroxide, hydroxylamine, and oxoammonium states. The radical and oxoammonium forms act as an efficient redox-pair via reversible one-electron redox reactions, while hydroxylamine and nitroxide radical forms do not constitute an effective redox-pair. This feature determines nitroxide radicals as perfect compounds for imaging of intracellular redox balance (92, 94). This redox cycle can be detected, using magnetic resonance imaging techniques as electron-paramagnetic resonance (EPR) spectroscopy and imaging (EPRI), as well as magnetic resonance imaging (MRI).

In the living systems, under physiological conditions, nitroxide radical (which possesses EPR and MRI contrast) undergos one-electron reduction to hydroxylamine (which is non-contrast form). Those reactions are reversible (93). The balance between oxidized and re-duced forms depends on the environment and especially from the oxygen availability, super-oxide production and status of the endogenous redox-pairs (e.g., NADH/NAD⁺, NADPH/NADP⁺, ascorbate/dehydroascorbate, etc.), which leads to reduction of nitroxide radical or oxidation of hydroxylamine. Thus, the ratio between radical and hydroxylamine forms directly depends on the redox status of the cells and tissues. Since only the radical form is characterized by EPR/MRI contrast, this could be used as a quantitative marker for the as-sessment of the redox status in biological objects in vitro and in vivo (91). Many excellent review articles have described this possibility (92, 95-98).

In conclusion, many researchers are focused on the differences between the redox status of normal and cancer cells and a variety of other pathologies, accompanied by high levels of ROS and disturbance of the intracellular redox homeostasis. Mitochondria are considered the main sources of intracellular ROS and as such they are essential for the functioning of normal and cancer cells. Recently, increasing studies are directed to detection of redox status of cells, tissues and body fluids. Development of various methodologies, including EPR spectroscopy and MRI in combination with nitroxide radicals as redox-responsive contrast substances, can improve the diagnosis of many diseases, characterized by mitochondrial dysfunction and re-dox imbalance.

Previous SectionNext Section

Acknowledgements

The study was partially supported by the Diversity grant (granted to R.B.).

Previous SectionNext Section

Footnotes

• This article is freely accessible online.

• Received July 31, 2017.
• Revision received August 9, 2017.
• Accepted August 10, 2017.

• Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved

Previous Section

 

References

1. ↵
    

   1. Trachootham D, 
   2. Lu W, 
   3. Ogasawara MA, 
   4. Valle NR-D, 
   5. Huang P

   : Redox regulation of cell survival. Antioxid Redox Signal 10: 1343-1374, 2008. 

   CrossRefMedlineGoogle Scholar
2. ↵
    

   1. Zhelev Z, 
   2. Bakalova R, 
   3. Aoki I, 
   4. Lazarova D, 
   5. Saga T

   : Imaging of superoxide generation in the dopaminergic area of the brain in Parkinson's disease, using mito-TEMPO. ACS Chem Neurosci 4: 1439-1445, 2013. 

   MedlineGoogle Scholar
3. ↵
    

   1. Giorgio M, 
   2. Trinei M, 
   3. Migliaccio E, 
   4. Pelicci PG

   : Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals? Nat Rev Mol Cell Biol 8: 722-728, 2007. 

   CrossRefMedlineGoogle Scholar
4. ↵
    

   1. Burgoyne JR, 
   2. Mongue-Din H, 
   3. Eaton P, 
   4. Shah AM

   : Redox signaling in cardiac physiology and pathology. Circ Res 111: 1091-1106, 2012. 

   Abstract/FREE Full Text
5. ↵
    

   1. Dröge W

   : Free radicals in the physiological control of cell function. Physiol Rev82: 47-95, 2002. 

   Abstract/FREE Full Text
6. ↵
    

   1. Dayal R, 
   2. Singh A, 
   3. Pandey A, 
   4. Mishra KP

   : Reactive oxygen species as mediator of tumor radiosensitivity. J Cancer Res Ther 10: 811-818, 2014. 

   Google Scholar
7. ↵
    

   1. Valko M, 
   2. Leibfritz D, 
   3. Moncol J, 
   4. Cronin MT, 
   5. Mazur M, 
   6. Telser J

   : Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39: 44-84, 2007. 

   CrossRefMedlineGoogle Scholar
8. 1. Weinberg F, 
   2. Chandel NS

   : Reactive oxygen species – development signaling regulates cancer. Cell Mol Life Sci 66: 3663-3673, 2009. 

   CrossRefMedlineGoogle Scholar
9. ↵
    

   1. Sena LA, 
   2. Chandel NS

   : Physiological roles of mitochondrial reactive oxygen species. Mol Cell 48: 158-167, 2012. 

   CrossRefMedlineGoogle Scholar
10. ↵
     

    1. Halliwell B

    : Biochemistry of oxidative stress. Biochem Soc Trans 35: 1147-1150, 2007. 

    Abstract/FREE Full Text
11. ↵
     

    1. Udensi UK, 
    2. Tchounwou PB

    : Dual effect of oxidative stress on leukemia cancer induction and treatment. J Exp Clin Cancer Res 33: 106, 2014. 

    Google Scholar
12. ↵
     

    1. Liou GY, 
    2. Storz P

    : Reactive oxygen species in cancer. Free Radic Res 44: 479-496, 2010. 

    CrossRefMedlineGoogle Scholar
13. ↵
     

    1. Jiang F, 
    2. Zhang Y, 
    3. Dusting GJ

    : NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol Rev 63: 218-242, 2011. 

    Abstract/FREE Full Text
14. ↵
     

    1. Ray PD, 
    2. Huang BW, 
    3. Tsuji Y

    : Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 24: 981-990, 2012. 

    CrossRefMedlineGoogle Scholar
15. 1. Karamalakova Y, 
    2. Chuttani K, 
    3. Sharma R, 
    4. Zheleva A, 
    5. Gadjeva V, 
    6. Mishra A

    : Biological eval‎uation of new potential anticancer agent for tumour imaging and radiotherapy by two methods: 99mTc-radiolabelling and EPR spectroscopy. Biotechnol Biotechnol Equip 28: 1172-1180, 2014. 

    Google Scholar
16. 1. Cobb CA, 
    2. Cole MP

    : Oxidative and nitrative stress in neurodegeneration. Neurobiol Dis 84: 4-21, 2015. 

    Google Scholar
17. 1. Rinnerthaler M, 
    2. Bischof J, 
    3. Streubel MK, 
    4. Trost A, 
    5. Richter K

    : Oxidative stress in aging human skin. Biomolecules 5: 545-589, 2015. 

    Google Scholar
18. 1. Tafani M, 
    2. Sansone L, 
    3. Limana F, 
    4. Arcangeli T, 
    5. Santis E, 
    6. Polese M, 
    7. Fini M, 
    8. Russo MA

    : The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression. Oxid Med Cell Longev 2016: 3907147, 2016.

    Google Scholar
19. 1. Nikolova G, 
    2. Karamalakova Y, 
    3. Kovacheva N, 
    4. Stanev S, 
    5. Zheleva A, 
    6. Gadjeva V

    : Protective effect of two essential oils isolated from Rosa damascena Mill. and Lavandula angustifolia Mill, and two classic antioxidants against L-dopa oxidative toxicity induced in healthy mice. Regul Toxicol Pharmacol 81: 1-7, 2016. 

    Google Scholar
20. ↵
     

    1. Rahman K

    : Studies on free radicals, antioxidants, and co-factors. Clin Interv Aging 2: 219-236, 2007. 

    MedlineGoogle Scholar
21. ↵
     

    1. Trachootham D, 
    2. Alexander J, 
    3. Huang P

    : Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 8: 579-581, 2009. 

    CrossRefMedlineGoogle Scholar
22. ↵
     

    1. Lowe SW, 
    2. Lin AW

    : Apoptosis in cancer. Carcinogenesis 21: 485-495, 2000.

    CrossRefMedlineGoogle Scholar
23. ↵
     

    1. Reuter S, 
    2. Gupta SC, 
    3. Chaturvedi MM, 
    4. Aggarwal BB

    : Oxidative stress, inflammation, and cancer: How are they linked? Free Radic Biol Med 49: 1603-1616, 2010.

    CrossRefMedlineGoogle Scholar
24. ↵
     

    1. Schafer FQ, 
    2. Buettner GR

    : Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30: 1191-1112, 2001. 

    CrossRefMedlineGoogle Scholar
25. ↵
     

    1. Han D, 
    2. Hanawa N, 
    3. Saberi B, 
    4. Kaplowitz N

    : Mechanisms of liver injury. III. Role of glutathione redox status in liver injury. Am J Physiol Gastrointest Liver Physiol 291: G1-G7, 2006. 

    Abstract/FREE Full Text
26. ↵
     

    1. Ivanova D, 
    2. Bakalova R, 
    3. Lazarova D, 
    4. Gadjeva V, 
    5. Zhelev Z

    : The impact of reactive oxygen species on anticancer therapeutic strategies. Adv Clin Exp Med 22: 899-908, 2013. 

    MedlineGoogle Scholar
27. 1. Solaini G, 
    2. Sgarbi G, 
    3. Baracca A

    : Oxidative phosphorylation in cancer cells. Biochim Biophys Acta 1807: 534-542, 2011. 

    CrossRefMedlineGoogle Scholar
28. ↵
     

    1. Zhelev Z, 
    2. Gadjeva V, 
    3. Aoki I, 
    4. Bakalova R, 
    5. Saga T

    : Cell-penetrating nitroxides as molecular sensors for imaging of cancer in vivo, based on tissue redox activity. Mo. Biosyst 8: 2733-2740, 2012. 

    Google Scholar
29. ↵
     

    1. Cui H, 
    2. Kong Y, 
    3. Zhang H

    : Oxidative stress, mitochondrial dysfunction, and aging. J Signal Transduct 2012: 646354, 2012. 

    CrossRefMedlineGoogle Scholar
30. ↵
     

    1. Zhou D, 
    2. Shao L, 
    3. Spitz DR

    : Reactive oxygen species in normal and tumor stem cells. Adv Cancer Res 122: 1-67, 2014. 

    CrossRefMedlineGoogle Scholar
31. ↵
     

    1. Mookerjee SA, 
    2. Divakaruni AS, 
    3. Jastroch M, 
    4. Brand MD

    : Mitochondrial uncoupling and lifespan. Mech Ageing Dev 131: 463-472, 2010. 

    CrossRefMedlineGoogle Scholar
32. ↵
     

    1. Handy DE, 
    2. Loscalzo J

    : Redox regulation of mitochondrial function. Antioxid Redox Signal 16: 1323-1367, 2012. 

    CrossRefMedlineGoogle Scholar
33. ↵
     

    1. Wallace DC

    : Mitochondria and cancer. Nat Rev Cancer 12: 685-698, 2012.

    CrossRefMedlineGoogle Scholar
34. ↵
     

    1. Arnould T, 
    2. Michel S, 
    3. Renard P

    : Mitochondria retrograde signaling and the UPRmt: where are we in mammals? Int J Mol Sci 16: 18224-18251, 2015. 

    CrossRefMedlineGoogle Scholar
35. ↵
     

    1. Apostolova N, 
    2. Victor VM

    : Molecular strategies for targeting antioxidants to mitochondria: Therapeutic Implications. Antioxid Redox Signal 22: 686-729, 2015.

    Google Scholar
36. ↵
     

    1. Kang J, 
    2. Pervaiz S

    : Mitochondria: redox metabolism and dysfunction. Biochem Res Int 2012: 896751, 2012. 

    CrossRefMedlineGoogle Scholar
37. ↵
     

    1. Nazarewicz RR, 
    2. Dikalova A, 
    3. Bikineyeva A, 
    4. Ivanov S, 
    5. Kirilyuk IA, 
    6. Grigor'ev IA, 
    7. Dikalov SI

    : Does scavenging of mitochondrial superoxide attenuate cancer prosurvival signaling pathways? Antioxid Redox Signal 19: 344-349, 2013.

    CrossRefMedlineGoogle Scholar
38. 1. Nikolova G, 
    2. Mancheva V

    : Analysis of the parameters of oxidative stress in patients with Parkinson's disease. Comp Clin Path 22: 151-155, 2013. 

    Google Scholar
39. 1. Mailloux RJ, 
    2. Jin X, 
    3. Willmorea WG

    : Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions. Redox Biol 2: 123-139, 2014.

    CrossRefMedlineGoogle Scholar
40. 1. Bakalova R, 
    2. Georgieva E, 
    3. Ivanova D, 
    4. Zhelev Z, 
    5. Aoki I, 
    6. Saga T

    : Magnetic resonance imaging of mitochondrial dysfunction and metabolic activity, accompanied by overproduction of superoxide. ACS Chem Neurosci 6: 1922-1929, 2015. 

    MedlineGoogle Scholar
41. ↵
     

    1. West AP, 
    2. Shadel GS, 
    3. Ghosh S

    : Mitochondria in innate immune response. Nat Rev Immunol 11: 389-402, 2011. 

    CrossRefMedlineGoogle Scholar
42. ↵
     

    1. Boland M, 
    2. Chourasia A, 
    3. Macleod K

    : Mitochondrial dysfunction in cancer. Front Oncol 3: 292, 2013. 

    CrossRefMedlineGoogle Scholar
43. ↵
     

    1. Frezza C

    : The role of mitochondria in the oncogenic signal transduction. Int J Biochem Cell Biol 48: 11-17, 2014. 

    CrossRefMedlineGoogle Scholar
44. ↵
     

    1. Marzetti E, 
    2. Csiszar A, 
    3. Dutta D, 
    4. Balagopal G, 
    5. Calvani R, 
    6. Leeuwenburgh C

    : Role of mitochondrial dysfunction and altered autophagy in cardiovascular aging and disease: from mechanisms to therapeutics. Mitochondria in cardiovascular physiology and disease. Am J Physiol Heart Circ Physiol 305: H459-H476, 2013.

    Abstract/FREE Full Text
45. ↵
     

    1. Tomkins AJ, 
    2. Burwell LS, 
    3. Digerness SB, 
    4. Zaragoza C, 
    5. Holman WL, 
    6. Brookes PS

    : Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition. Biochim Biophys Acta 1762: 223-231, 2006.

    CrossRefMedlineGoogle Scholar
46. ↵
     

    1. Burton GJ, 
    2. Jauniaux E

    : Oxidative stress. Best Pract Res Clin Obstet Gynaecol 25: 287-299, 2011. 

    CrossRefMedlineGoogle Scholar
47. 1. Cade WT

    : Diabetes-related microvascular and macrovascular diseases in the physical therapy setting. Phys Ther 88: 1322-1335, 2008. 

    Abstract/FREE Full Text
48. 1. Circu ML, 
    2. Aw TY

    : Reactive oxygen species, cellular redox systems and apoptosis. Free Radical Biol Med 48: 749-762, 2010. 

    CrossRefMedlineGoogle Scholar
49. ↵
     

    1. Closa D, 
    2. Folch-Puy E

    : Oxygen free radicals and the systemic inflammatory response. IUBMB Life 56: 185-191, 2004. 

    CrossRefMedlineGoogle Scholar
50. ↵
     

    1. Gao L, 
    2. Laude K, 
    3. Cai H

    : Mitochondrial pathophysiology, reactive oxygen species, and cardiovascular diseases. Vet Clin North Am Small Anim Pract 38: 137-155, 2008. 

    CrossRefMedlineGoogle Scholar
51. ↵
     

    1. Stowe DF, 
    2. Camara AK

    : Mitochondrial reactive oxygen species production in excitable cells: modulators of mitochondrial and cell function. Antioxid Redox Signal 11: 1373-1414, 2009. 

    CrossRefMedlineGoogle Scholar
52. 1. Winkler BS, 
    2. Arnold MJ, 
    3. Brassell MA, 
    4. Puro DG

    : Energy metabolism in human retinal Müller cells. Invest Ophthalmol Vis Sci 41: 3183-3190, 2000.

    Abstract/FREE Full Text
53. 1. Li L, 
    2. Prabhakaran K, 
    3. Mills EM, 
    4. Borowitz JL, 
    5. Isom GE

    : Enhancement of cyanide-induced mitochondrial dysfunction and cortical cell necrosis by uncoupling protein-2. Toxicol Sci 86: 116-124, 2005. 

    CrossRefMedlineGoogle Scholar
54. 1. Li L, 
    2. Bu S, 
    3. Bäckström T, 
    4. Landström M, 
    5. Ulmsten U, 
    6. Fu X

    : Induction of apoptosis and G₂/M arrest by 2-methoxyestradiol in human cervical cancer HeLaS3 cells. Anticancer Res 24: 873-880, 2004. 

    Abstract/FREE Full Text
55. ↵
     

    1. Pelicano H, 
    2. Feng L, 
    3. Zhou Y, 
    4. Carew JS, 
    5. Hileman EO, 
    6. Plunkett W, 
    7. Keating MJ, 
    8. Huang P

    : Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J Biol Chem 278: 37832-37839, 2003. 

    Abstract/FREE Full Text
56. 1. Moysés DN, 
    2. Barrabin H

    : Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca²⁺ accumulation. Biochim Biophys Acta 1656: 96-103, 2004. 

    MedlineGoogle Scholar
57. 1. Chen Q, 
    2. Vazquez EJ, 
    3. Moghaddas S, 
    4. Hoppel CL, 
    5. Lesnefsky EJ

    : Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem 278: 36027-36031, 2003. 

    Abstract/FREE Full Text
58. 1. Shchepina LA, 
    2. Pletjushkina OY, 
    3. Avetisyan AV, 
    4. Bakeeva LE, 
    5. Fetisova EK, 
    6. Izyumov DS, 
    7. Saprunova VB, 
    8. Vyssokikh MY, 
    9. Chernyak BV, 
    10. Skulachev VP

    : Oligomycin, inhibitor of the F0 part of H⁺-ATP-synthase, suppresses the TNF-induced apoptosis. Oncogene21: 8149-8157, 2002. 

    CrossRefMedlineGoogle Scholar
59. ↵
     

    1. Wyatt CN, 
    2. Buckler KJ

    : The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells. J Physiol 556: 175-191, 2004. 

    CrossRefMedlineGoogle Scholar
60. ↵
     

    1. Kalbácová M, 
    2. Vrbacký M, 
    3. Drahota Z, 
    4. Melková Z

    : Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry. Cytometry A 52: 110-116, 2003.

    MedlineGoogle Scholar
61. ↵
     

    1. Zheng G, 
    2. Lyu J, 
    3. Huang J, 
    4. Xiang D, 
    5. Xie M, 
    6. Zeng Q

    : Experimental treatments for mitochondrial dysfunction in sepsis: A narrative review. J Res Med Sci 20: 185-195, 2015. 

    Google Scholar
62. ↵
     

    1. Hargreaves IP, 
    2. Duncan AJ, 
    3. Wu L, 
    4. Agrawal A, 
    5. Land JM, 
    6. Heales SJ

    : Inhibition of mitochondrial complex IV leads to secondary loss complex II-III activity: implications for the pathogenesis and treatment of mitochondrial encephalomyopathies. Mitochondrion 7: 284-287, 2007. 

    CrossRefMedlineGoogle Scholar
63. 1. Srinivasan S, 
    2. Avadhani NG

    : Cytochrome c oxidase dysfunction in oxidative stress. Free Radic Biol Med 53: 1252-1263, 2012. 

    CrossRefMedlineGoogle Scholar
64. ↵
     

    1. Brand MD, 
    2. Nicholls DG

    : Assessing mitochondrial dysfunction in cells. Biochem J435: 297-312, 2011. 

    Abstract/FREE Full Text
65. ↵
     

    1. Leavesley HB, 
    2. Li L, 
    3. Prabhakaran K, 
    4. Borowitz JL, 
    5. Isom GE

    : Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity. Toxicol Sci 101: 101-111, 2008. 

    CrossRefMedlineGoogle Scholar
66. ↵
     

    1. Sherer TB, 
    2. Betarbet R, 
    3. Kim JH, 
    4. Greenamyre JT

    : Selective microglial activation in the rat rotenone model of Parkinson's disease. Neurosci Lett 341: 87-90, 2003.

    CrossRefMedlineGoogle Scholar
67. ↵
     

    1. Radad K, 
    2. Rausch WD, 
    3. Gille G

    : Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration. Neurochem Int 49: 379-386, 2006. 

    CrossRefMedlineGoogle Scholar
68. ↵
     

    1. Jung SY, 
    2. Lee KW, 
    3. Choi SM, 
    4. Yang EJ

    : Bee venom protects against rotenone-induced cell death in nsc34 motor neuron cells. Toxins (Basel) 7: 3715-3726, 2015. 

    Google Scholar