Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

Isoliquiritigenin isolated from Glycyrrhiza uralensis protects neuronal cells against glutamate-induced mitochondrial dysfunction

Glutamate-mediated excitotoxicity, which is associated with reactive oxygen species (ROS), is hypothesized to be a major contributor to pathological cell death in the mammalian central nervous system, and to be involved in many acute and chronic brain diseases. Here, we showed that isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis (Gu), one of the most frequently prescribed oriental herbal medicines, protected HT22 hippocampal neuronal cells from glutamate-induced oxidative stress. In addition, we clarified the molecular mechanisms by which it protects against glutamate-induced neuronal cell death. ISL reversed glutamate-induced ROS production and mitochondrial depolarization, as well as glutamate-induced changes in expression of the apoptotic regulators Bcl-2 and Bax. Pretreatment of HT22 cells with ISL suppresses the release of apoptosis-inducing factor from mitochondria into the cytosol. Taken together, our results suggest that ISL may protect against mitochondrial dysfunction by limiting glutamate-induced oxidative stress. In conclusion, our results demonstrated that ISL isolated from Gu has protective effects against glutamate-induced mitochondrial damage and hippocampal neuronal cell death. We expect ISL to be useful in the development of drugs to prevent or treat neurodegenerative diseases.     

Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.     

Cofilin activation mediates Bax translocation to mitochondria during excitotoxic neuronal death

During excitotoxic neuronal death, Bax translocates to the mitochondria where it plays an important role by contributing to the release of proapoptotic factors. However, how Bax translocates to the mitochondria during excitotoxicity remains poorly understood. Herein, our data suggest the presence of a novel signalling mechanism by which NMDA receptor stimulation promotes Bax translocation. This signalling pathway is triggered by dephosphorylation of cofilin. Once dephosphorylated, cofilin might interact physically with Bax acting as a carrier for it, translocating it to the mitochondria, where it contributes to mitochondrial membrane despolarization, permeabilization and to the release of apoptotic factors, thus leading to neuronal death. Lack-of-function studies indicate that only the Slingshot family of phosphatases, more specifically the enzyme Slingshot 1L phosphatase, but not cronophin participates in the cofilin activation process during excitotoxicity. Indeed, cofilin-mediated Bax translocation seems to be a key event in excitotoxic neuronal death as knock down of either cofilin or Slingshot 1L phosphatase has a marked neuroprotective effect on NMDA-mediated neuronal death. This novel biochemical pathway may therefore be a good target to develop future therapeutic molecules for neurodegenerative diseases.     

Mitochondria regulation in ferroptosis

Ferroptosis is recognized as a new form of regulated cell death which is initiated by severe lipid peroxidation relying on reactive oxygen species (ROS) generation and iron overload. This iron-dependent cell death manifests evident morphological, biochemical and genetic differences from other forms of regulated cell death, such as apoptosis, autophagy, necrosis and pyroptosis. Ferroptosis was primarily characterized by condensed mitochondrial membrane densities and smaller volume than normal mitochondria, as well as the diminished or vanished of mitochondria crista and outer membrane ruptured. Mitochondria take the center role in iron metabolism, as well as substance and energy metabolism as it’s the major organelle in iron utilization, catabolic and anabolic pathways. Interference of key regulators of mitochondrial lipid metabolism (e.g., ASCF2 and CS), iron homeostasis (e.g., ferritin, mitoferrin1/2 and NEET proteins), glutamine metabolism and other signaling pathways make a difference to ferroptotic sensitivity. Targeted induction of ferroptosis was also considered as a potential therapeutic strategy to some oxidative stress diseases, including neurodegenerative disorders, ischemia-reperfusion injury, traumatic spinal cord injury. However, the pertinence between mitochondria and ferroptosis is still in dispute. Here we systematic elucidate the morphological characteristics and metabolic regulation of mitochondria in the regulation of ferroptosis.     

Hydrogen sulfide guards myoblasts from ferroptosis by inhibiting ALOX12 acetylation

Recognized as a novel and important gasotransmitter, hydrogen sulfide (H₂S) is widely present in various tissues and organs. Cystathionine gamma-lyase (CSE)-derived H₂S has been shown to regulate oxidative stress and lipid metabolism. The aim of the present study is to examine the role of H₂S in ferroptosis and lipid peroxidation in mouse myoblasts and skeletal muscles. Ferroptosis agonist RSL3 inhibited the expressions of Gpx4 and reduced CSE/H₂S signaling, which lead to increased oxidative stress, lipid peroxidation, and ferroptotic cell death. In addition, ferroptosis antagonist ferrostatin-1 (Fer-1) up-regulated the expression of CSE, scavenged the generation of reactive oxygen species (ROS) and lipid peroxidation, and improved cell viability. Exogenously applied NaHS was also able to block RSL3-induced ferroptotic cell death. Neither RSL3 nor H₂S affected cell apoptosis. Furthermore, H₂S reversed RSL3-induced Drp1 expression and mitochondrial damage, which lead to abnormal lipid metabolism as evidenced by altered expressions of ACSL4, FAS, ACC and CPT1 as well as higher acetyl-CoA contents in both cytoplasm and mitochondria. RSL3 promoted the protein expression and acetylation of ALOX12, a key protein in initiating membrane phospholipid oxidation, while the addition of NaHS attenuated ALOX12 acetylation and protected from membrane lipid peroxidation. Moreover, we observed that CSE deficiency alters the expressions of ferroptosis and lipid peroxidation-related proteins and enhances global protein acetylation in mouse skeletal muscles under aging or injury conditions. These results indicate that downregulation of CSE/H₂S signaling would contribute to mitochondrial damage, abnormal lipid metabolism, membrane lipid peroxidation, and ferroptotic cell death. CSE/H₂S system can be a target for preventing ferroptosis in skeletal muscle.     

Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress

Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases.     

Plasma-activated medium induces ferroptosis by depleting FSP1 in human lung cancer cells

Cold atmospheric plasma (CAP) that generates reactive oxygen species (ROS) has received considerable scientific attentions as a new type of anticancer. In particular, an indirect treatment method of inducing cancer cell death through plasma-activated medium (PAM), rather than direct plasma treatment has been well established. Although various cell death pathways such as apoptosis, necroptosis, and autophagy have been suggested to be involved in PAM-induced cell death, the involvement of ferroptosis, another type of cell death regulated by lipid ROS is largely unknown. This study reports, that PAM promotes cell death via ferroptosis in human lung cancer cells, and PAM increases intracellular and lipid ROS, thereby resulting in mitochondrial dysfunction. The treatment of cells with N-acetylcysteine, an ROS scavenging agent, or ferrostatin-1, a ferroptosis inhibitor, protects cells against PAM-induced cell death. Interestingly, ferroptosis suppressor protein 1 (FSP1) is downregulated upon PAM treatment. Furthermore, the treatment of cells with iFSP1, an inhibitor of FSP1, further enhances PAM-induced ferroptosis. Finally, this study demonstrates that PAM inhibits tumor growth in a xenograft model with an increase in 4-hydroxynoneal and PTGS2, a byproduct of lipid peroxidation, and a decrease in FSP1 expression. This study will provide new insights into the underlying mechanism and therapeutic strategies of PAM-mediated cancer treatment.Subject terms: Non-small-cell lung cancer, Drug development     

Redox signaling (cross-talk) from and to mitochondria involves mitochondrial pores and reactive oxygen species

This review highlights the important role of redox signaling between mitochondria and NADPH oxidases. Besides the definition and general importance of redox signaling, the cross-talk between mitochondrial and Nox-derived reactive oxygen species (ROS) is discussed on the basis of 4 different examples. In the first model, angiotensin-II is discussed as a trigger for NADPH oxidase activation with subsequent ROS-dependent opening of mitochondrial ATP-sensitive potassium channels leading to depolarization of mitochondrial membrane potential followed by mitochondrial ROS formation and respiratory dysfunction. This concept was supported by observations that ethidium bromide-induced mitochondrial damage suppressed angiotensin-II-dependent increase in Nox1 and oxidative stress. In another example hypoxia was used as a stimulator of mitochondrial ROS formation and by using pharmacological and genetic inhibitors, a role of mitochondrial ROS for the induction of NADPH oxidase via PKC? was demonstrated. The third model was based on cell death by serum withdrawal that promotes the production of ROS in human 293T cells by stimulating both the mitochondria and Nox1. By superior molecular biological methods the authors showed that mitochondria were responsible for the fast onset of ROS formation followed by a slower but long-lasting oxidative stress condition based on the activation of an NADPH oxidase (Nox1) in response to the fast mitochondrial ROS formation. Finally, a cross-talk between mitochondria and NADPH oxidases (Nox2) was shown in nitroglycerin-induced tolerance involving the mitochondrial permeability transition pore and ATP-sensitive potassium channels. The use of these redox signaling pathways as pharmacological targets is briefly discussed.     

Mitochondrial Uncoupling as a Therapeutic Target Following Neuronal Injury

Mitochondrial dysfunction is a prominent feature of excitotoxic insult and mitochondria are known to play a pivotal role in neuronal cell survival and death following injury. Following neuronal injury there is a well-documented increase in cytosolic Ca(2+), reactive oxygen species (ROS) production and oxidative damage. In vitro studies have demonstrated these events are dependent on mitochondrial Ca(2+) cycling and that a reduction in membrane potential is sufficient to reduce excitotoxic cell death. This concept has gained additional support from experiments demonstrating that the overexpression of endogenous mitochondrial uncoupling proteins (UCP), which decrease the mitochondrial membrane potential, decreases cell death following oxidative stress. Our group has demonstrated that upregulation of UCP activity can reduce excitotoxic-mediated ROS production and cell death whereas a reduction in UCP levels increases susceptibility to neuronal injury. These findings raise the possibility that mitochondrial uncoupling could be a potential novel treatment for acute CNS injuries.     

Regulatory pathways and drugs associated with ferroptosis in tumors

Ferroptosis is a type of cell death that depends on iron and reactive oxygen species (ROS). The accumulation of iron and lipid peroxidation primarily initiates oxidative membrane damage during ferroptosis. The core molecular mechanism of ferroptosis includes the regulation of oxidation and the balance between damage and antioxidant defense. Tumor cells usually contain a large amount of H₂O₂, and ferrous/iron ions will react with excessive H₂O₂ in cells to produce hydroxyl radicals and induce ferroptosis in tumor cells. Here, we reviewed the latest studies on the regulation of ferroptosis in tumor cells and introduced the tumor-related signaling pathways of ferroptosis. We paid particular attention to the role of noncoding RNA, nanomaterials, the role of drugs, and targeted treatment using ferroptosis drugs for mediating the ferroptosis process in tumor cells. Finally, we discussed the currently unresolved problems and future research directions for ferroptosis in tumor cells and the prospects of this emerging field. Therefore, we have attempted to provide a reference for further understanding of the pathogenesis of ferroptosis and proposed new targets for cancer treatment.Subject terms: Cancer, Cell death, Non-coding RNAs     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stabilization of mitochondrial function by tetramethylpyrazine protects against kainate-induced oxidative lesions in the rat hippocampus

Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Reactive oxygen species (ROS) are crucial to Ca²⁺-mediated effects of glutamate receptor activation leading to neuronal degeneration. Tetramethylpyrazine (TMP) is a principal ingredient of Ligusticum wallichi Franchat (a Chinese herb), used for treatment of cardiovascular and cerebrovascular ischemic diseases. However, its protection against oxidative brain injury associated with excessive activation of glutamate receptors is unknown. In this study, we demonstrate TMP neuroprotection against kainate-induced excitotoxicity in vitro and in vivo. We found that TMP could partly alleviate kainate-induced status epilepticus in rats and prevented and rescued neuronal loss in the hippocampal CA3 but not the CA1 region. The partial prevention and rescue of neuronal loss by TMP were attributable to the preservation of the structural and functional integrity of mitochondria, evidenced by maintaining the mitochondrial membrane potential, ATP production, and complex I and III activities. Stabilization of mitochondrial function was linked to the observation that TMP could function as a reductant/antioxidant to quench ROS, block lipid peroxidation, and protect enzymatic antioxidants such as glutathione peroxidase and glutathione reductase. These results suggest that TMP may protect against oxidative brain injury by stabilization of mitochondrial function through quenching of ROS.     

Mitochondria bridge HIF signaling and ferroptosis blockage in acute kidney injury

Ferroptosis, a form of regulated cell death, plays an important role in acute kidney injury (AKI). Previous studies have shown that prolyl hydroxylase domain protein (PHD) inhibitors that activate HIF signaling provide strong protection against AKI, which is characterized by marked cell death. However, the relationship between PHD inhibition/HIF signaling and ferroptosis in AKI has not been elucidated. Here, we review recent studies to explore the issue. First, we will review the literature concerning the functions of HIF in promoting mitophagy, suppressing mitochondrial respiration and modulating redox homeostasis. Second, we will describe the current understanding of ferroptosis and its role in AKI, particularly from the perspective of mitochondrial dysfunction. Finally, we will discuss the possibility that mitochondria link PHD inhibition/HIF signaling and ferroptosis in AKI. In conclusion, we propose that HIF may protect renal cells against ferroptosis in AKI by reducing mitochondrial oxidative stress and damage.Subject terms: Cell biology, Kidney diseases     

Iron homeostasis and oxidative stress: An intimate relationship

Iron is a transition metal and essential constituent of almost all living cells and organisms. As component of various metalloproteins it is involved in critical biochemical processes such as transport of oxygen in tissues, electron transfer reactions during respiration in mitochondria, synthesis and repair of DNA, metabolism of xenobiotics, etc. However, when present in excess within cells and tissues, iron disrupts redox homeostasis and catalyzes the propagation of reactive oxygen species (ROS), leading to oxidative stress. ROS are critical for physiological signaling pathways, but oxidative stress is associated with tissue injury and disease. At the cellular level, oxidative stress may lead to ferroptosis, an iron-dependent form of cell death. In this review, we focus on the intimate relationship between iron metabolism and oxidative stress in health and disease. We discuss aspects of redox- and iron-mediated signaling, toxicity, ferroptotic cell death, homeostatic pathways and pathophysiological implications.     

Benefits of Iron Chelators in the Treatment of Parkinson’s Disease

As a novel discovered regulated cell death pattern, ferroptosis has been associated with the development of Parkinson’s disease (PD) and has attracted widespread attention. Nevertheless, the relationship between ferroptosis and PD pathogenesis is still unclear. This study aims to investigate the effect of iron overload on dopaminergic (DA) neurons and its correlation with ferroptosis. Here we use nerve growth factor (NGF) induced PC12 cells which are derived from pheochromocytoma of the rat adrenal to establish a classical PD in vitro model. We found significantly decreased cell viability in NGF-PC12 cell under ammonium ferric citrate (FAC) administration. Moreover, excessive intracellular iron ions induced the increase of (reactive oxygen species) ROS release as well as the decrease of mitochondrial membrane potential in PC12-NGF cells. In addition, we also found that overloaded iron can activate cell apoptosis and ferroptosis pathways, which led to cell death. Furthermore, MPP-induced PD cells were characterized by mitochondrial shrinkage, decreased expression of glutathione peroxidase 4 (Gpx4) and ferritin heavy chain (FTH1), and increased divalent metal transporter (DMT1) and transferrin receptor 1 (TfR1) expression level. In contrast, Lip-1 and DFO increased the expression level of GPX4 and FTH1 compared to MPP-induced PD cell. In conclusion, we indicated that overloaded intracellular iron contributes to neurons death via apoptosis and ferroptosis pathways, while DFO, an iron chelator, can inhibit ferroptosis in order to protect the neurons in vitro.

     

SirT3 Regulates the Mitochondrial Unfolded Protein Response

The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPR^mt) has been reported recently. So far, the UPR^mt has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPR^mt. Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.     

Mitochondrial Dysfunction and Reactive Oxygen Species in Excitotoxicity and Apoptosis: Implications for the Pathogenesis of Neurodegenerative Diseases

In recent years we have witnessed a major interest in the study of the role of mitochondria, not only as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca²⁺ homeostasis and endogenous producers of reactive oxygen species (ROS). Interestingly, the mitochondria have been also implicated as central executioners of cell death. Increased mitochondrial Ca²⁺ overload as a result of excitotoxicity has been associated with the generation of superoxide and may induce the release of proapoptotic mitochondrial proteins, proceeding through DNA fragmentation/condensation and culminating in cell demise by apoptosis and/or necrosis. In addition, these processes have been implicated in the pathogenesis of many neurodegenerative diseases, which share several features of cell death: selective brain areas undergo neurodegeneration, involving mitochondrial dysfunction (mitochondrial complexes are affected), loss of intracellular Ca²⁺ homeostasis, excitotoxicity, and the extracellular or intracellular accumulation of insoluble protein aggregates in the brain.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.     

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants — the “Skulachev-ion” derivatives

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1–2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of “bad” mitochondria in cell physiology is discussed.