On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid

Glycyrrhetinic acid (GE), the aglycone of glycyrrhizic acid, a triterpene glycoside which represents one of the main constituents of licorice root, induces an oxidative stress in liver mitochondria responsible for the induction of membrane permeability transition. In fact, GE, by interacting with the mitochondrial respiratory chain, generates hydrogen peroxide which in turn oxidizes critical thiol groups and endogenous pyridine nucleotides leading to the opening of the transition pore. Most likely the reactive group of GE is the carbonyl oxygen in C-11 which, by interacting mainly with a Fe/S centre of mitochondrial complex I, generates an oxygen-centered radical responsible for the pro-oxidant action.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 microM, GE prevents oxidative stress and MPT induced by supraphysiological Ca2+ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca2+. GE also inhibits Ca2+ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca2+ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Oxidative stress is responsible for mitochondrial permeability transition induction by salicylate in liver mitochondria

The interaction of salicylate with the respiratory chain of liver mitochondria generates hydrogen peroxide and, most probably, other reactive oxygen species, which in turn oxidize thiol groups and glutathione. This oxidative stress, confirmed by the prevention of action by antioxidant agents, leads to the induction of the mitochondrial permeability transition in the presence of Ca2+. This phenomenon induces further increase of oxidative damage resulting in impairment of oxidative phosphorylation and beta-oxidation, cardinal features of Reye's syndrome in the liver. Mitochondrial permeability transition induction also induces the release of cytochrome c and apoptotic inducing factor from mitochondria, suggesting that salicylate also behaves as a pro-apoptotic agent. The reactive group of salicylate for inducing oxidative stress is the hydroxyl group which, by interacting with a Fe-S cluster of mitochondrial Complex I, the so-called N-2(Fe-S) center, produces reactive oxygen species.     

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the omega 6 to omega 3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the omega 6 to omega 3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondrial membrane-associated enzymes.     

Role of short-chain hydroxyacyl CoA dehydrogenases in SCHAD deficiency

Short-chain hydroxyacyl CoA dehydrogenase deficiency is an ill-defined, severe pediatric disorder of mitochondrial fatty acid β-oxidation of short-chain hydroxyacyl CoAs. To understand the relative contributions of the two known short-chain hydroxyacyl CoA dehydrogenases (HADH) tissue biopsies of six distinct family individuals were analyzed and kinetic parameters were compared. Steady-state kinetic constants for HADH 1 and HADH 2 suggest that type 1 is the major enzyme involved in mitochondrial β-oxidation of short-chain hydroxyacyl-CoAs. Two patients are heterozygous carriers of a HADH 1 polymorphism, whereas no mutation is detected in the HADH 2 gene of all patients. The data suggest that protein interactions rather than HADH mutations are responsible for the disease phenotype. Pull-down experiments of recombinant HADH 1 and 2 with human mitochondrial extracts reveal two proteins interacting with HADH 1, one of which was identified as glutamate dehydrogenase. This association provides a possible link between fatty acid metabolism and the hyperinsulinism/hyperammonia syndrome.     

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/β-catenin signaling

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/β-catenin pathway assessed by phosphorylation of GSK-3β. Over-expressing of constitutively active GSK-3β (caGSK-3β) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3β over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.     

The mitochondrial permeability transition in toxic, hypoxic and reperfusion injury

Opening of a non-specific, high conductance permeability transition pore or megachannel in the inner mitochondrial membrane causes onset of the mitochondrial permeability transition, which is characterized by mitochondrial swelling, depolarization and uncoupling. Inducers of the permeability transition include Ca²⁺, oxidant stress and a permissive pH greater than 7.0. Blockers include cyclosporin A, trifluoperazine and pH < 7. Using laser scanning confocal microscopy, we developed techniques to visualize onset of the mitochondrial permeability transition in situ in living cells. In untreated cells, the permeability transition pore is continuously closed and does not 'flicker' open. By contrast, the pore opens in liver and heart cells after exposure to oxidant chemicals, calcium ionophore, hypoxia and ischemia/reperfusion, causing mitochondrial uncoupling and aggravation of ATP depletion. In injury to hepatocytes from tert-butylhydroperoxide, an analog of lipid hydroperoxides generated during oxidative stress, onset of the mitochondrial permeability transition is preceded by oxidation of mitochondrial pyridine nucleotides, mitochondrial generation of oxygen radicals and an increase of mitochondrial Ca²⁺, all inducers of the mitochondrial permeability transition. In ischemia, the acidosis of anaerobic metabolism protects strongly against cell death. During reperfusion, recovery of pH to normal levels is a stress that actually precipitates cell killing. Onset of the mitochondrial permeability transition may be responsible, in part, for this pH-dependent injury, or pH paradox. The mitochondrial permeability transition may also be responsible for a variety of pathological phenomena. In particular, the mitochondrial permeability transition may underlie Reye's syndrome and Reye's-like drug toxicities. In conclusion, multiple mechanisms contribute to cell injury after hypoxia, ischemia/reperfusion and toxic chemicals, but a common final pathway leading to acute cellular nec rosis may be ATP depletion after mitochondrial failure. One important mechanism causing mitochondrial failure is the mitochondrial permeability transition, which both uncouples oxidative phosphorylation and accelerates ATP hydrolysis. Interventions that block this pH-dependent phenomenon protect against onset of cell death. (Mol Cell Biochem 174: 159–165, 1997)     

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Geraniol Protects Against the Protein and Oxidative Stress Induced by Rotenone in an In Vitro Model of Parkinson’s Disease

Dysfunction of autophagy, mitochondrial dynamics and endoplasmic reticulum (ER) stress are currently considered as major contributing factors in the pathogenesis of Parkinson’s disease (PD). Accumulation of oxidatively damaged cytoplasmic organelles and unfolded proteins in the lumen of the ER causes ER stress and it is associated with dopaminergic cell death in PD. Rotenone is a pesticide that selectively kills dopaminergic neurons by a variety of mechanism, has been implicated in PD. Geraniol (GE; 3,7-dimethylocta-trans-2,6-dien-1-ol) is an acyclic monoterpene alcohol occurring in the essential oils of several aromatic plants. In this study, we investigated the protective effect of GE on rotenone-induced mitochondrial dysfunction dependent oxidative stress leads to cell death in SK-N-SH cells. In addition, we assessed the involvement of GE on rotenone-induced dysfunction in autophagy machinery via α-synuclein accumulation induced ER stress. We found that pre-treatment of GE enhanced cell viability, ameliorated intracellular redox, preserved mitochondrial membrane potential and improves the level of mitochondrial complex-1 in rotenone treated SK-N-SH cells. Furthermore, GE diminishes autophagy flux by reduced autophagy markers, and decreases ER stress by reducing α-synuclein expression in SK-N-SH cells. Our results demonstrate that GE possess its neuroprotective effect via reduced rotenone-induced oxidative stress by enhanced antioxidant status and maintain mitochondrial function. Furthermore, GE reduced ER stress and improved autophagy flux in the neuroblastomal SK-N-SH cells. The present study could suggest that GE a novel therapeutic avenue for clinical intervention in neurodegenerative diseases especially for PD.     

Urate causes the human polymorphonuclear leukocyte to secrete superoxide.

The human polymorphonuclear leukocyte generates O2-. and H2O2 when it is treated with uric acid. A transition metal catalyzed reaction between O2-. and H2O2 can give the hydroxyl radical and myeloperoxidase forms hypochlorous acid from H2O2 and chloride. Therefore, the uric acid-induced secretion of oxidants may be responsible for a large part of the inflammation associated with gout.     

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria

Previously mildronate, an aza-butyrobetaine derivative, was shown to be a cytoprotective drug, through its mechanism of action of inhibition of carnitine palmitoyltransferase-1, thus protecting mitochondria from long-chain fatty acid accumulation and subsequent damage. Recently in an azidothymidine (AZT)-induced cardiotoxicity model in vivo (in mice), we have found mildronate's ability of protecting heart tissue from nuclear factor kappaB abnormal expression. Preliminary data also demonstrate cerebro- and hepatoprotecting properties of mildronate in AZT-toxicity models. We suggest that mildronate may target its action predominantly to mitochondria. The present study in isolated rat liver mitochondria was designed to clarify mitochondrial targets for mildronate by using AZT as a model compound. The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT-induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronate's regulatory action. The results showed that mildronate protected mitochondria from AZT-induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT-caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronate's effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions.     

Suppression of Mitochondrial Oxidative Phosphorylation and TCA Enzymes in Discrete Brain Regions of Mice Exposed to High Fluoride: Amelioration by Panax ginseng (Ginseng) and Lagerstroemia speciosa (Banaba) Extracts

Chronic fluoride intoxication results in pathophysiological complications pertaining to soft tissues, called non-skeletal fluorosis. This study examined whether fluoride-induced alterations in selected parameters that are indicative of mitochondrial dysfunction accompany the toxic effects of fluoride in discrete brain regions in vivo and also explored the possibility of treatment with Ginseng (GE) and Banaba (BLE) either alone or with their co-exposure which is capable of reversing parameters indicative of fluoride-induced impairments in mitochondrial function. Swiss mice, Mus musculus, were given 270 ppm fluoride (600 ppm NaF) in their drinking water for 30 days, while continuing the fluoride exposure, toxicated animals were given differential doses (50–250 mg/kg body wt) of phytoextracts through oral gavage for 2 weeks. Discrete brain regions separated from dissected animals to perform biochemical assessments. Disturbances in mitochondrial enzyme complexes (I-IV) and decrements in TCA enzymes (ICDH, SDH, and aconitase) were noted in discrete brain regions upon F exposure, suggesting mitochondrial dysfunction. In addition, a significant reduction in oxidative stress indices with increased MDA content as well as decrease in reduced glutathione content and increases in catalase and SOD enzyme activity suggests the involvement of severe oxidative stress affecting the mitochondrial function(s). Treatment with either GE or BLE reversed F-induced alterations in augmenting the suppressed complex enzymes followed by TCA enzymes and oxidative stress indices in a dose independent manner. However, the co-exposure of GE and BLE at a dose of 150 mg/kgbw appeared to restore mitochondrial functioning. These results provide in vivo evidence supporting the hypothesis that fluoride induces impairments in mitochondrial function, which can be reversed by treatment with GE and BLE as well their co-exposure at 150 mg/kgbw.     

Mitochondrial fatty acid synthesis and respiration

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.     

Genome editing to generate nonfoam-forming sake yeast strains

ABSTRACT

Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1?/awa1? deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1?/awa1? in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding.     

Glutamine in the Pathogenesis of Hepatic Encephalopathy: The Trojan Horse Hypothesis Revisited

Hepatic encephalopathy (HE) is major neuropsychiatric disorder occurring in patients with severe liver disease and ammonia is generally considered to represent the major toxin responsible for this condition. Ammonia in brain is chiefly metabolized (“detoxified”) to glutamine in astrocytes due to predominant localization of glutamine synthetase in these cells. While glutamine has long been considered innocuous, a deleterious role more recently has been attributed to this amino acid. This article reviews the mechanisms by which glutamine contributes to the pathogenesis of HE, how glutamine is transported into mitochondria and subsequently hydrolyzed leading to high levels of ammonia, the latter triggering oxidative and nitrative stress, the mitochondrial permeability transition and mitochondrial injury, a sequence of events we have collectively termed as the Trojan horse hypothesis of hepatic encephalopathy.     

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H₂O₂ generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H₂O₂ and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca²⁺. Instead, 1 mM agmatine generates larger amounts of H₂O₂ than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca²⁺-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.     

Oxidation of mitochondrial peroxiredoxin 3 during the initiation of receptor-mediated apoptosis

It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-alpha. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.