Mol Cell Biochem (2007) 305:235–253 DOI 10.1007/s11010-007-9520-8
Mitochondria: a hub of redox activities and cellular distress control Poonam Kakkar Æ B. K. Singh
Received: 22 January 2007 / Accepted: 16 May 2007 / Published online: 12 June 2007 Springer Science+Business Media B.V. 2007
Abstract In their reductionist approach in unraveling phenomena inside the cell, scientists in recent times have focused attention to mitochondria. An organelle with peculiar evolutionary history and organization, it is turning out to be an important cell survival switch. Besides controlling bioenergetics of a cell it also has its own genetic machinery which codes 37 genes. It is a major source of generation of reactive oxygen species, acts as a safety device against toxic increases of cytosolic Ca2+ and its membrane permeability transition is a critical control point in cell death. Redox status of mitochondria is important in combating oxidative stress and maintaining membrane permeability. Importance of mitochondria in deciding the response of cell to multiplicity of physiological and genetic stresses, inter-organelle communication, and ultimate cell survival is constantly being unraveled and discussed in this review. Mitochondrial events involved in apoptosis and necrotic cell death, such as activation of Bcl-2 family proteins, formation of permeability transition pore, release of cytochrome c and apoptosis inducing factors, activation of caspase cascade, and ultimate cell death is the focus of attention not only for cell biologists, but also for toxicologists in unraveling stress responses. Mutations caused by ROS to mitochondrial DNA, its inability to repair it completely and creation of a vicious cycle of mutations along with role of Bcl-2 family genes and proteins has been implicated in many diseases where mitochondrial
P. Kakkar (&) B. K. Singh Herbal Research Section, Industrial Toxicology Research Centre, P.O. Box-80, M G Marg, Lucknow 226 001, India e-mail: [email protected]
dysfunctions play a key role. New therapeutic approaches toward targeting low molecular weight compounds to mitochondria, including antioxidants is a step toward nipping the stress in the bud. Keywords Mitochondria Redox regulation Electron transport Reactive oxygen species Calcium homeostasis Mitochondrial diseases
Introduction Mitochondria are double membrane organelles, and form an integral part of all eukaryotic cells and are sometimes described as ‘‘cellular power plants,’’ because they fulfill our energy needs by converting organic materials into energy . Mitochondrion although discovered before 1900s, research on this organelle did not gain momentum probably due to ignorance about far-reaching implications and applications of the discoveries in mitochondrial metabolism and genome (mtDNA). It took some time to accumulate sufficient knowledge to comprehend the future importance of mitochondrial research. Two recent developments have brought mitochondria in the limelight. First, starting in the late 1980s, genetic basis for a large number and variety of ‘mitochondrial diseases’ has been gradually revealed. There are also indications that Parkinson’s disease, Diabetes mellitus, possibly Alzheimer’s disease, and even aging in general are influenced in time of onset and severity by mitochondrial deficiencies or dysfunction. Second, mitochondria have been shown to play a central role in apoptosis, with wide-ranging implications in cancer research, senescence, and finally death of an organism .
Evolutionary history of mitochondria Mitochondria are thought to have evolved from a bacterial (prokaryote) progenitor. In fact, the whole evolution of sophisticated life forms has been dependent on this evolutionary step. The chloroplasts consolidate energy from the sun and produce organic food, and the mitochondria use those fuels to generate energy-rich biochemical compounds. The very first evidence of their independent bacterial origin is the presence of mitochondrial DNA (mtDNA) inside, a simple circular genome that has structural characteristics very similar to the circular DNA of primitive bacteria . Research of the past 30 years has largely strengthened the hypothesis of mitochondria as endosymbionts of a primitive eukaryote . The sequential endo-symbiont theory postulates that proto-eukaryotic cell without mitochondria evolved first, which subsequently captured a proteobacterium by endocytosis. After establishing a symbiotic relationship, the loss of redundant genes and the transfer of genes from the bacterium to the nucleus lead to the currently observed distribution of genes between the two genomes [5, 6]. Table 1 summarizes some of the landmarks in research on mitochondria. Some of the earliest studies of mammalian hybrid and cybrid cell lines had revealed that mitochondria from one mammalian species are generally incompatible with the nuclear genome of another. One can speculate that the subunits of electron transport complexes encoded by the nuclear genome must be compatible with those encoded by mtDNA. Recent studies have shown that chimpanzee and gorilla mitochondria can be functional when introduced into human cells which are lacking mtDNA, but orangutan mitochondria are not fully functional [7, 8]. Such studies strengthen the belief that nuclear and mitochondrial coding sequences had to evolve in a manner that assured retention of functional interactions of respective subunits of electron transport chain (ETC) complexes .
Energy metabolism in mitochondria In the living cell, mitochondria contribute in a number of different processes. Among these are ATP synthesis by oxidative phosphorylation, the production of reactive oxygen species (ROS), Ca++ uptake and release, production of NADPH, synthesis of DNA, RNA, protein, DNA repair, thermogenesis (in some cases), and metabolic pathways . Of these, the major mitochondrial event involved in cell life is ATP synthesis by oxidative phosphorylation [10–12]. Most important achievements in biochemistry and bioenergetics have been in the study of mitochondria with the discovery of Kreb’s cycle, electron transport chain and
Mol Cell Biochem (2007) 305:235–253 Table 1 Landmarks in mitochondrial research Major events
Discovery of mitochondria inside the cell and known them as bacteria
Prior to 1900
Biochemistry of Urea and TCA cycle
Electron microscopic description of mitochondria
Mechanism of fatty acid oxidation Mitochondria as a site for respiration and oxidative phosphorylation Discovery of cytoplasmic inheritance in yeast Analysis of respiratory chain machinery Discovery of DNA inside the mitochondria
Fractionation and characterization of major complexes of electron transport chain and ATP synthase Define mobile carriers, cytochrome c and ubiquinone Peter D. Mitchell proposed the chemiosmotic hypothesis
Mammalian mitochondrial DNA sequenced completely and all the genes identified
Elucidation of RNA transcripts and mRNAs Identification of translation products In vitro mitochondrial protein import Identified mtDNA replication Molecular identification of mitochondrial disorders Plant mitochondrial DNA sequenced completely
1990s till date
Introduction of mtDNA in yeast by microprojectile Participation of mitochondria in apoptosis (recognition of bcl-2, cyt c, permeability transition) Crystal structure of complex III, complex IV, F1ATPsynthase
oxidative phosphorylation. Peter Mitchell received Nobel Prize in 1978 for his proposed chemiosmotic hypothesis . Mitochondria are the site of most ATP production. Therefore, the tissues having high metabolic rates like muscles, brain, liver, and heart contain relatively highest numbers of mitochondria. This organelle contains four well-defined compartments, an inner membrane, an outer membrane, a matrix space enclosed by the inner membrane, and an inter-membrane space between the inner and outer membranes . Majority of the estimated 1,000 different mitochondrial proteins are encoded by nuclear DNA, translated in the cytoplasm and transported into the mitochondria. The production of ATP occurs through respiratory chain, which is divided into multi-enzyme complexes (proteins) embedded in the inner mitochondrial membrane . They are (Fig. 1): (1) complex I: NADHCoQ reductase, (2) complex II: succinate-CoQ reductase, (3) complex III: reduced CoQ-cytochrome c reductase, (4) complex IV: cytochrome c oxidase, and (5) complex V: ATP synthase.
Mol Cell Biochem (2007) 305:235–253
Oxidative phosphorylation occurs in the inner membrane and requires an electron transport chain to generate an electrochemical proton gradient, two key transport systems for the entry of ADP and Pi into the matrix space, and an ATP synthase complex, called F0F1, that binds the ADP and Pi and utilizes the bulk of the gradient to drive the synthesis and release of ATP. The remaining part of the gradient is used, in part, to drive the transport of Pi and ADP into the matrix space and to drive the net synthesis of NADPH via a transmembrane enzyme called transhydrogenase, which is not part of the oxidative phosphorylation apparatus. Complex I (NADH dehydrogenase) initiates the process of electron flow by oxidizing NADH to NAD+. Electrons then flow sequentially through complex III (bc1 complex), cytochrome c, and finally through complex IV (cytochrome oxidase), where bound oxygen is reduced to water. During the process of electron flow from NADH to molecular oxygen, each of the three complexes (I, III, and IV) catalyzes the translocation of protons across the mitochondrial inner membrane. Extensive data collected over the past four decades; including recent crystal structures of complex III [16, 17] and complex IV [18, 19] implicate the involvement of quite different chemistries for generation of electrochemical proton gradient. For example, complex III catalyzes a unique cycle known as the ‘‘Q cycle’’ in which the proton carrier is a lipid called coenzyme Q or ubiquinone. In contrast, cytochrome oxidase is believed to translocate protons via one (or more) proton pathway(s) that involve specific amino acid functional groups [18, 20]. When intact mitochondria carry out oxidative phosphorylation, the F0 base piece transmits the energy from the electrochemical proton gradient (generated by the electron
transport chain) to the F1 unit, promoting the synthesis and release of ATP. Oxidative stress and redox regulation in mitochondria Concentration of reactive oxygen species (ROS) and reactive nitrogen species (RNS), when cross a certain threshold level, overwhelm the antioxidant defenses, resulting into oxidative stress. In a healthy cell, excessive harmful ROS/RNS are decomposed by protective antioxidant machinery / detoxification processes. ROS/RNS homeostasis is important (Fig. 2) as certain types of ROS (such as H2O2) and RNS (particularly nitric oxide, NO) are membrane permeable and diffuse across it. Those can be elevated to non-harmful (optimum) levels under specific physiological conditions, thus playing physiological signaling roles. H2O2 acts in concert with thiol redox systems such as glutathione and thioredoxin, an important example being apoptosis or programmed cell death. The time scales of these processes are important, whereas ROS/RNS reactions are fast, response of the cell/organism, i.e., variations in ROS/RNS homeostasis are much slower. Elevated ROS/RNS levels over short periods (less than minutes) can manage to initiate protein expression and other regulations, yet they cannot be completed within these short timings . ROS include radical species (a free, i.e., unpaired, electron-containing species), such as superoxide (O 2 ), hydroperoxyl radical (HO2 ), hydroxyl (OH ), carbonate (CO 3 ), peroxyl (RO2 ), and alkoxyl (RO ) radicals. Some non-radical species are also recognized as ROS, like H2O2, singlet oxygen (1O2) and other compounds . Similarly, RNS include radical species such as nitric oxide ( NO), nitrogen dioxide ( NO2 ), in addition to non-radical peroxynitrite OONO–, N2O3, N-nitrosoamines, S-nitrosothiols, nitrosated fatty acids, and other compounds [21, 23, 24]. According to Schafer and Buettner , a group of reactive sulfur species (RSS) deserves its own definition. ROS/RNS sources inside mitochondria Major sources of ROS
Fig. 1 Schematic representation of various complexes involved in oxidative phosphorylation (OXPHOS): Complexes I–V are embedded in inner mitochondrial membrane. Pumping of protons from matrix to the inter-membrane space creates a membrane potential which in turn is harnessed to synthesize high energy phosphate molecule, ATP
Mitochondria use approximately 90% of the consumed O2 for oxidative phosphorylation and ATP synthesis, thus the proteins involved in the mitochondrial electron transport chain are probable sites for ROS generation. Mitochondrial electron transport chain generates superoxide (O 2 ) as an unavoidable by-product and primary ROS at two complexes, complexes I and III [26–33]. There is a continuing debate on the exact sites of the production of O 2 and on their relative contribution in vivo.
Mol Cell Biochem (2007) 305:235–253
Fig. 2 Oxidative stress (ROS/ RNS) generation and antioxidant machinery. One electron transfer to the O2 leads to generation of superoxide radical which after dismutation in the presence of SOD generates H2O2. It can then give rise to OH radical through Fenton’s or Heber-Weiss reaction which is highly reactive and can lead to peroxidative decomposition of PUFA, or converted to H2O by glutathione peroxidase in the process converting GSH to GSSG and back by glutathione reductase. NO can combine with O 2 to give rise to peroxynitrite which is highly reactive
At complex I (NADH coenzyme Q reductase) the sites are thought to be the iron-sulfur centers [34, 35] or the ‘active site flavin’ . Based on inhibitor studies in heart mitochondria, cytochrome b was proposed to be the site of superoxide production rather than ubisemiquinone at complex O [37, 38]. However, numerous studies have demonstrated that the double inhibition of complex III by inhibitors antimycin A (acts at site Qi, and blocks electron flow from cytochrome b560 to ubiquinone or ubisemiquinone) and myxothiazol (acts at site Qo, inhibiting electron flow from ubiquinol to FeS center) results in consistent decreases in superoxide production . This strongly supports the idea that unstable ubisemiquinone molecules, and not the b cytochromes, are the major sites of superoxide production at complex III. Complex I and complex III are widely thought to produce superoxide on the matrix side of the membrane . Furthermore, Han et al.  demonstrated superoxide production by liver mitoplasts in the presence of antimycin A, supporting the idea that superoxide is released into the inter-membrane space. Significant increase in hydrogen peroxide production with the addition of superoxide dismutase to the antimycin-A treated mitochondria was interpreted as additional evidence that superoxide may be released into the inter-membrane space. Cadenas et al. [28, 42] have revealed that the production of superoxide by complex III is released to both the matrix and cytosolic sides of the mitochondria which is approximately 80% and 20%, respectively. The relative contributions of complexes I and III to ROS production appear to be dependent on types of tissues, species and experimental conditions . The rate of ROS production is affected substantially by the mitochondrial metabolic state. Under state 4, when oxygen consumption is low the proton-motive force is high and complexes of the electron transport chain are in reduced
states, then superoxide production is highest. A common misconception is that the production of ROS is highest when the rate of oxygen consumption is highest, with the notion that superoxide production is a fixed proportion of oxygen consumption. The major factor underlying the proton motive force-dependent production of superoxide is the occupancy of the outer Q-site of complex III with the semiquinone anion . This is the case when ATP demand is low and proton motive force is high. Moreover, it has been known for more than 30 years that upon the addition of ADP, and the resulting transition to state 3 respiration, superoxide production diminishes dramatically [43–45]. Since, the proportion of oxygen consumed that emerges as ROS is highest under nonphosphorylating, rather than phosphorylating conditions, most determinations of mitochondrial ROS production are made under state 4 conditions . Lipid peroxidation Lipid peroxidation is known to generate new radical source initiated by the highly reactive radicals . Non-enzymatic lipid peroxidation starts by formation of carboncentered radicals (RC R) at the ‘‘iso-prostanoid’’ units of unsaturated fatty acid chains of phospholipids (PL) and other lipid components . Oxidation of RC R with O2 provides peroxyl radicals (LOO ) which react, for example, with the nearby polyunsaturated fatty acid (PUFA)-side chain (or the phospholipase A2 (PLA2), cleaves phospholipids side chains releasing free PUFAs that in turn react with LOO [49–53]. These reactions yield hydroperoxide side chains (LOOH, i.e., PLOOH, phospholipids hydroperoxide) or free fatty acid hydroperoxides (FAOOH) and other RC R. PLOOH may be later cleaved by PLA2 to yield again FAOOH. It is interesting to note that mitochondria
Mol Cell Biochem (2007) 305:235–253
contain both Ca2+-insensitive isoforms of PLA2 [49–53] as well as Ca2+-dependent PLA2 isoform activated by superoxide . All PLA2 isoforms cleave FAOOHs from the phospholipids side chains. Overall, lipoperoxidation is a self-propagating reaction within the membrane . In the presence of Fe2+, alkoxyradicals (RO ) are formed by Fenton chemistry and react again with a nearby PUFA. Notably, 4-hydroxy-2-nonenal is formed during peroxidation of b-6 PUFA side chains (e.g., linoleic, arachidonic acid) by spontaneous cleavage of PLOOH in the presence of transition-metal ions , which may also be a signaling molecule [51, 55, 56]. (i) LH þ OH ! L þ H2 O ðiiÞ L þ O2 ! LOO ðiiiÞ LH þ LOO ! L þ LOOH
Sources of RNS Physiologically important, nitric oxide ( NO) is a highly diffusible free radical, formed by conversion of arginine to citrulline via a family of nitric oxide synthase (NOS) isoenzymes, or by a specific form of mitochondrial nitric oxide synthase (mtNOS) in certain tissues, namely liver, brain, thymus, and heart [57, 58]. Non-mitochondrial NOS are represented by the constitutively expressed neuronal nNOS and endothelial eNOS as well as by inducible iNOS, expressed during inflammation. NO reversibly (and competitively with O2) inhibits the mitochondrial cytochrome c oxidase, which seems to be its primary regulatory role in mitochondria . Palacois-Callender et al.  showed that O 2 production in the respiratory chain is enhanced by the modified redox state of cytochrome c oxidase, in addition, NO helps to maintain relatively higher respiration at very low presence of O2, which in fact means physiological levels of O2. Such regulatory property is even more important during hypoxia. NO may also inhibit Complex I by several mechanisms . (i) OH þ NO 2 ! NO2 þ OH
ðiiÞ 2NO þ O2 ! 2 NO2 ðiiiÞ NO þ NO2 ! N2 O3 ðivÞ NO þ O 2 ! ONOO
HO can oxidize nitrite to nitrogen dioxide ( NO2 ), whereas NO also reacts directly with oxygen to produce NO2 , which can then combine with NO giving N2O3. NO2 can oxidize a wide range of biomolecules, including PUFAs (lipid nitration). N2O3 can add nitrosate group (NO+) to amines, yielding N-nitroso-amines followed by thiols, giving S-nitrosothiols, (e.g., S-nitrosoglutathione) or to proteins (e.g., S-nitrosoalbumin). The NO+ group is transferable between S-nitrosothiols. A significant RNS, peroxynitrite (OONO–) forms when NO combines with O 2 in a diffusion controlled manner [21, 23, 24] which can further react with CO2, lipids, etc., to yield OH . Thus, these reactions may represent an even more powerful source of OH than Fenton chemistry . Peroxynitrite can readily oxidize (and nitrate) various biomolecules, i.e., the –SH groups of cysteine, GSH, and the S atom of methionine, ascorbate, the purine and pyrimidine bases of DNA, and protein tyrosine residues yielding nitrotyrosine. Peroxynitrite itself initiates lipid peroxidation. It has been experimentally seen that when the formation rates for NO and O 2 increased, lipid peroxidation is increased, whereas when the former exceeds the latter, lipid peroxidation is suppressed . In mitochondria, peroxynitrite can inhibit Complexes I, II, and IV, the ATP synthase, aconitase, creatine kinase, and even MnSOD . Under conditions of increased cellular or mitochondrial NO formation and increased O 2 release, the formed peroxynitrite alters mitochondrial oxidative phosphorylation and Ca2+ homeostasis phenomena associated with diseases and aging. Notably, peroxynitritedependent induction of Ca2+ efflux from mitochondria has been observed [60–62]. This may be related to the formation of ‘‘permeability transition pore,’’ which could be responsible for a Ca2+ efflux leading to subsequent release of cytochrome c and other factors, and to the initiation of apoptosis.
Calcium dynamics and active oxygen species Major oxidative metabolic processes of the cell take place inside the mitochondria that are highly susceptible to the attack of active oxygen species produced by oxidative metabolism due to the presence of many electron carriers and PUFA rich membranes . Disturbance of intracellular ion concentration occurs as an early response to injurious stimuli in many cells and tissues . Mitochondria have the highest Ca2+ accumulation capacity among the various cell organelles . It has also been seen that any toxic stress mediated increase in cellular Ca2+ concentration modulates calcium dynamics in mitochondria. The rapid accumulation of Ca2+ through potential
dependent uniport mechanism of Ca2+ uptake by mitochondria, removes excess Ca2+ from the cytosol bringing it down to levels from where it can be taken care of by the endoplasmic reticulum . Kappus and Comporti [67, 68] acknowledged that active oxygen species cause peroxidative decomposition of PUFA component of lipid bilayer of biological membranes followed by membrane damage as well as permeability changes. A variety of chemically different pro-oxidants cause Ca2+ release from the mitochondria via a route which is physiologically relevant. When the released Ca2+ is excessively cycled by mitochondria, they are damaged . This shows the way to uncoupling, decrease in ATP supply and a decreased ability of mitochondria to retain Ca2+. During CaCl2 induced swelling of mitochondria the peroxidative decomposition of membrane lipids also gets enhanced [70, 71]. In 1993, research in author’s lab revealed that  many respiratory uncouplers like, dinitrophenol, antimycin-A, rotenone, sodium azide, potassium cyanide in the absence of Ca2+ had no significant effect on swelling of mitochondria but during the Ca2+ mediated swelling, these respiratory uncouplers caused significant changes. The uncouplers and inhibitors of oxidative phosphorylation were found to reduce the swelling caused in the presence of Ca2+. Respiratory chain blockers like KCN and sodium azide inhibit oxygen uptake thereby reducing the oxygen radical formation where as antimycin and rotenone decrease hydrogen peroxide transfer and increase NADH causing reduction in swelling. Castilho et al.  and Kakkar et al.  have reported tertiary-butylhydroperoxide mediated oxidative damage to the mitochondria in the presence of Ca2+. Oxidation of critical thiol groups followed by protein aggregation has also been reported by Wyatt et al. . A few studies showed that the in vitro swelling of mitochondria caused by peroxidative factors was retarded by use of antioxidants .
Antioxidant machinery inside and outside mitochondria ROS detoxification At neutral pH, inside the cellular system, superoxide anion (O 2 ) is a moderately stable free radical. Its toxicity is principally based on generation of further reactive species, which are then able to attack intracellular biomolecules, as discussed earlier. However, its conjugated acid HO2 ; the hydroperoxyl radical, is extremely reactive . O 2 in the matrix is converted to H2O2 by matrix MnSOD , while O 2 released to the intramembrane space is partly dismutated by the presence of inter-membrane space CuZn-SOD [30, 79]. Any residual O 2 which diffuses into the cytosol
Mol Cell Biochem (2007) 305:235–253
is similarly converted by the cytosolic CuZnSOD. If any mitochondrial O 2 can reach the extra cellular space, it is detoxified by extra cellular CuZn-SOD. Once H2O2 is produced, it can easily penetrate through membranes, due to its interchanged nature and poor reactivity. On the other hand, H2O2 may be converted to the most reactive hydroxyl radical OH by transition-metal catalyzed reactions [54, 80], most notably by Fe2+. In addition, reactions of peroxynitrite with CO2, lipids, etc., also yield the OH . Significantly, inactivation of matrix aconitase by oxidation of its iron-sulfur cluster and concomitant release of Fe2+, results in OH production  which is an extremely reactive species affecting most biomolecules in a short time. Hence scavenging of this radical is an unlikely mechanism of action for any antioxidant in vivo . Once the OH arises, for example in the matrix, it usually does not cross the inner membrane, even though it is hydrophobic. Much lower reactivity of O 2 and H2O2 means that they can diffuse away from their sites of formation and their toxicity is further determined by the availability and distribution of metal ions, that together with H2O2 give rise to OH in different cellular locations. The OH may further react with bicarbonate yielding the very reactive carbonate radical anion (CO 3 ) under physiological conditions. These very reactive radicals, OH ; HO2 ; and CO 3 attack proteins  and DNA, and also induce non-enzymatic lipid peroxidation [24, 48], while when produced in the matrix side, they attack mitochondrial DNA . Redox regulation Fundamentally, there are two types of major redox systems present outside the mitochondria. These are glutathionebased system, including glutathione-S transferase and the thioredoxin system, including peroxiredoxins [80, 84, 85]. Since the concentration of glutathione (GSSG/GSH) is so much higher in comparison to any other system (intracellular concentration of thioredoxin is 100–1,000-fold lower than glutathione), the GSH/GSSG pool within the cell dominates the intracellular redox environment and comprises the principal redox system of the cell. Thus H2O2 and other ROS are degraded by a few selenium dependent glutathione peroxidases (GPx, three isoforms) which, with glutathione, convert H2O2 to water and also convert free FAOOHs to their corresponding hydroxyl acids (FAOH). The fourth isoform of GPx (GPx4) specifically acts in membranes on hydroperoxy groups of peroxidized phospholipid side chains and on cholesterol hydroperoxides [86–88]. Finally, GSSG is reduced to GSH by glutathione reductase (GR). NADPH is required as cofactor for GR. Mitochondria and some organelles have their own GSH pool independent of the cytosolic system.
Mol Cell Biochem (2007) 305:235–253
In the cell system most of the free glutathione is present in the reduced form as GSH rather then GSSG, because of GSSG reactivity with protein thiol groups leading to formation of mixed disulfides in a process called protein S-thiolation. It subsequently causes protein inhibition. Since GSH prevails over its oxidized GSSG form, any fractional ROS detoxification leading to oxidation of a limited amount of GSH will affect the redox status of the cell, which also regulates important cell regulatory pathways . Thus GSH depletion sensitizes cells to apoptotic stimuli. Glutathione is also present in extra cellular compartments. The main source of plasma glutathione is the liver, which constantly excretes GSH. Apart from its role as a cofactor for GPx family enzymes, GSH itself has scavenging properties and reacts with OH ; HOCl, RO ; RO2 ; carbon-centered radicals, peroxynitrite, and O2. This gives thiyl (GS) radical that potentially generates O2 and other ROS. It thus has the capacity to increase total ROS inside the cytosol. Thus, SOD co-operates with GSH in helping to remove free radicals. Other systems degrading H2O2 and ROS are proteins of thioredoxin family [89, 90], acting with the thioredoxindependent peroxide reductases, i.e., peroxiredoxins  and a family of selenium-independent glutathione-S transferases, GST . The GSTs catalyze GSH-dependent reduction of both PLOOH and FAOOH, and their respective aldehydes. Thioredoxin, is especially concentrated in the endoplasmic reticulum. It is a rather thiol-specific antioxidant that reduces disulfide bridges of proteins subjected to oxidative stress. But, whereas glutathione forms inter-molecular disulfides, thioredoxin produces mostly intra-molecular ones and its system has different substrate preferences. It is also very important to know that NADPH is a source of reducing equivalents for both thioredoxin and glutathione systems. NADPH with thioredoxin reductase reduces 12 kDa proteins of the thioredoxin family, which together with GSH or peroxiredoxins maintain protein thiols in the reduced state . Another detoxification enzyme, catalase, can contribute to ROS detoxification. The activity of catalase, converting H2O2 to water, is restricted in most tissues to peroxisomes. Remarkably, heart mitochondria are the only mitochondria that also contain catalase .
In mitochondria, matrix glutathione represents 10–15% of the total glutathione in liver . Surprisingly, its capacity is estimated to be only 15% of the rate of H2O2 production , therefore, H2O2 diffuses out of the mitochondrial matrix. Since the enzymes for GSH synthesis are not present in the matrix, GSH must be imported into mitochondria from the cytosol, by a putative carrier. The original work by Martensson et al.  showed that at external GSH levels