Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3–4 months of age) female Sprague–Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction.     

Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation

Brain seizure activity is characterised by intense activation of mitochondrial oxidative phosphorylation. This stimulation of oxidative phosphorylation is in the low magnesium model of seizure-like events accompanied by substantial increase in formation of reactive oxygen species (ROS). However, it has remained unclear which ROS-generating sites can be attributed to this phenomenon. Here, we report stimulatory effects of calcium ions and uncouplers, mimicking mitochondrial activation, on ROS generation of isolated rat and mouse brain mitochondria. Since these stimulatory effects were visible with superoxide sensitive dyes, but with hydrogen peroxide sensitive dyes only in the additional presence of SOD, we conclude that the complex redox properties of the ‘Qo’ center at respiratory chain complex III are very likely responsible for these observations. In accordance with this hypothesis redox titrations of the superoxide production of antimycin-inhibited submitochondrial particles with the succinate/fumarate redox couple confirmed for brain tissue a bell-shaped dependency with a maximal superoxide production rate at + 10 mV (pH = 7.4). This reflects the complex redox properties of a semiquinone species which is the direct electron donor for oxygen reduction in complex III-dependent superoxide production. Therefore, we conclude that under conditions of increased energy load the complex III site can contribute to superoxide production of brain mitochondria, which might be relevant for epilepsy-related seizure activity.     

Low hydrogen peroxide production in mitochondria of the long‐lived Arctica islandica: underlying mechanisms for slow aging

The observation of an inverse relationship between lifespan and mitochondrial H₂O₂ production rate would represent strong evidence for the disputed oxidative stress theory of aging. Studies on this subject using invertebrates are surprisingly lacking, despite their significance in both taxonomic richness and biomass. Bivalve mollusks represent an interesting taxonomic group to challenge this relationship. They are exposed to environmental constraints such as microbial H₂S, anoxia/reoxygenation, and temperature variations known to elicit oxidative stress. Their mitochondrial electron transport system is also connected to an alternative oxidase that might improve their ability to modulate reactive oxygen species (ROS) yield. Here, we compared H₂O₂ production rates in isolated mantle mitochondria between the longest‐living metazoan—the bivalve Arctica islandica—and two taxonomically related species of comparable size. In an attempt to test mechanisms previously proposed to account for a reduction of ROS production in long‐lived species, we compared oxygen consumption of isolated mitochondria and enzymatic activity of different complexes of the electron transport system in the two species with the greatest difference in longevity. We found that A. islandica mitochondria produced significantly less H₂O₂ than those of the two short‐lived species in nearly all conditions of mitochondrial respiration tested, including forward, reverse, and convergent electron flow. Alternative oxidase activity does not seem to explain these differences. However, our data suggest that reduced complex I and III activity can contribute to the lower ROS production of A. islandica mitochondria, in accordance with previous studies. We further propose that a lower complex II activity could also be involved.     

A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (<Emphasis Type="Italic">Hemiscyllum ocellatum</Emphasis>)

Hypoxia and warm ischemia are primary concerns in ischemic heart disease and transplant and trauma. Hypoxia impacts tissue ATP supply and can induce mitochondrial dysfunction that elevates reactive species release. The epaulette shark, Hemiscyllum ocellatum, is remarkably tolerant of severe hypoxia at temperatures up to 34°C, and therefore provides a valuable model to study warm hypoxia tolerance. Mitochondrial function was tested in saponin permeabilised ventricle fibres using high-resolution respirometry coupled with purpose-built fluorospectrometers. Ventricular mitochondrial function, stability and reactive species production of the epaulette shark was compared with that of the hypoxia-sensitive shovelnose ray, Aptychotrema rostrata. Fibres were prepared from each species acclimated to normoxic water conditions, or following a 2 h, acute hypoxic exposure at levels representing 40% of each species’ critical oxygen tension. Although mitochondrial respiratory fluxes for normoxia-acclimated animals were similar for both species, reactive species production in the epaulette shark was approximately half that of the shovelnose ray under normoxic conditions, even when normalised to tissue oxidative phosphorylation flux. The hypoxia-sensitive shovelnose ray halved oxidative phosphorylation flux and cytochrome c oxidase flux was depressed by 34% following hypoxic stress. In contrast, oxidative phosphorylation flux of the epaulette shark ventricular fibres isolated from acute hypoxia exposed the animals remained similar to those from normoxia-acclimated animals. However, uncoupling of respiration revealed depressed electron transport systems in both species following hypoxia exposure. Overall, the epaulette shark ventricular mitochondria showed greater oxidative phosphorylation stability and lower reactive species outputs with hypoxic exposure, and this may protect cardiac bioenergetic function in hypoxic tropical waters.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species

Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.     

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H₂O₂ release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (α-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1–0.2% of O₂ consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.     

Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes

VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.     

Energy,ageing, fidelity and sex: oocyte mitochondrial DNA as a protected genetic template

Oxidative phosphorylation couples ATP synthesis to respiratory electron transport. In eukaryotes, this coupling occurs in mitochondria, which carry DNA. Respiratory electron transport in the presence of molecular oxygen generates free radicals, reactive oxygen species (ROS), which are mutagenic. In animals, mutational damage to mitochondrial DNA therefore accumulates within the lifespan of the individual. Fertilization generally requires motility of one gamete, and motility requires ATP. It has been proposed that oxidative phosphorylation is nevertheless absent in the special case of quiescent, template mitochondria, that these remain sequestered in oocytes and female germ lines and that oocyte mitochondrial DNA is thus protected from damage, but evidence to support that view has hitherto been lacking. Here we show that female gametes of Aurelia aurita, the common jellyfish, do not transcribe mitochondrial DNA, lack electron transport, and produce no free radicals. In contrast, male gametes actively transcribe mitochondrial genes for respiratory chain components and produce ROS. Electron microscopy shows that this functional division of labour between sperm and egg is accompanied by contrasting mitochondrial morphology. We suggest that mitochondrial anisogamy underlies division of any animal species into two sexes with complementary roles in sexual reproduction. We predict that quiescent oocyte mitochondria contain DNA as an unexpressed template that avoids mutational accumulation by being transmitted through the female germ line. The active descendants of oocyte mitochondria perform oxidative phosphorylation in somatic cells and in male gametes of each new generation, and the mutations that they accumulated are not inherited. We propose that the avoidance of ROS-dependent mutation is the evolutionary pressure underlying maternal mitochondrial inheritance and the developmental origin of the female germ line.     

Reactive oxygen species and mitochondrial diseases

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the mitochondria as byproducts of normal cell respiration. The interrelationship between ROS and mitochondria suggests shared pathogenic mechanisms in mitochondrial and ROS-related diseases. Defects in oxidative phosphorylation can increase ROS production, whereas ROS-mediated damage to biomolecules can have direct effects on the components of the electron transport system. Here, we review the molecular mechanisms of ROS production and damage, as well as the existing evidence of mitochondrial ROS involvement in human diseases.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content

Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe²⁺ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the Q_O site of complex III.     

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ₁₀), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H₂O₂)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ₁₀ can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ₁₀ not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ₁₀ prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ₁₀ preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ₁₀ may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

Mechanisms of vanadate-induced cellular toxicity: role of cellular glutathione and NADPH

Increased production of reactive oxygen species (ROS) by the mitochondrion has been implicated in the pathogenesis of numerous liver diseases. However, the exact sites of ROS production within liver mitochondria and the electron transport chain are still uncertain. To determine the sites of ROS generation in liver mitochondria we evaluated the ability of a variety of mitochondrial respiratory inhibitors to alter the steady state levels of ROS generated within the intact hepatocyte and in isolated mitochondria. Treatment with myxothiazol alone at concentrations that significantly inhibit respiration dramatically increased the steady-state levels of ROS in hepatocytes. Similar results were also observed in isolated mitochondria oxidizing succinate. Coincubation with antimycin or rotenone had no effect on myxothiazol-induced ROS levels. Myxothiazol stimulation of ROS was mitochondrial in origin as demonstrated by the colocalization of MitoTracker Red and dichlorofluorescein staining using confocal microscopy. Furthermore, diphenyliodonium, an inhibitor that blocks electron flow through the flavin mononucleotide of mitochondrial complex I and other flavoenzymes, significantly attenuated the myxothiazol-induced increase in hepatocyte ROS levels. Together, these data suggest that in addition to the ubiquinone-cytochrome bc(1) complex of complex III, several of the flavin-containing enzymes or iron-sulfur centers within the mitochondrial electron transport chain should also be considered sites of superoxide generation in liver mitochondria.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo

Background  

The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria.