cytochrome-c-t and Mitochondrial-Diseases

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.

Topics: Apoptosis; Cell Respiration; Cytochromes c; Eukaryotic Cells; Gene Expression Regulation; Humans; Mitochondrial Diseases

Mitochondrial dysfunction is a global term used in the context of "unhealthy" mitochondria. In practical terms, mitochondria are extremely complex and highly adaptive in structure, chemical and enzymatic composition, subcellular distribution and functional interaction with other components of cells. Consequently, altered mitochondrial properties that are used in experimental studies as measures of mitochondrial dysfunction often provide little or no distinction between adaptive and maladaptive changes. This is especially a problem in terms of generation of oxidant species by mitochondria, wherein increased generation of superoxide anion radical (O(2*)(-)) or hydrogen peroxide (H(2)O(2)) is often considered synonymously with mitochondrial dysfunction. However, these oxidative species are signaling molecules in normal physiology so that a change in production or abundance is not a good criterion for mitochondrial dysfunction. In this review, we consider generation of reactive electrophiles and consequent modification of mitochondrial proteins as a means to define mitochondrial dysfunction. Accumulated evidence indicates that 4-hydroxynonenal (HNE) modification of proteins reflects mitochondrial dysfunction and provides an operational criterion for experimental definition of mitochondrial dysfunction. Improved means to detect and quantify mitochondrial HNE-protein adduct formation could allow its use for environmental healthrisk assessment. Furthermore, application of improved mass spectrometry-based proteomic methods will lead to further understanding of the critical targets contributing to disease risk.

Topics: Aldehydes; Cardiolipins; Cytochromes c; Glutathione; Humans; Hydrogen Peroxide; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Oxidative Stress; Sulfhydryl Compounds; Superoxides

To present the recent findings obtained in clinical and experimental studies examining microcirculatory alterations in sepsis, their link to mitochondrial dysfunction, and current knowledge regarding the impact of these alterations on the outcome of septic patients.. Interlinked by a mutual cascade effect and driven by the host-pathogen interaction, microcirculatory and mitochondrial functions are impaired during sepsis. Mitochondrial respiration seems to evolve during the course of sepsis, demonstrating a change from reversible to irreversible inhibition. The spatiotemporal heterogeneity of microcirculatory and mitochondrial dysfunction suggests that these processes may be compartmentalized. Although a causal relationship between mitochondrial and microcirculatory dysfunction and organ failure in sepsis is supported by an increasing number of studies, adaptive processes have also emerged as part of microcirculatory and mitochondrial alterations. Treatments for improving or preserving microcirculatory, mitochondrial function, or both seem to yield a better outcome in patients.. Even though there is evidence that microcirculatory and mitochondrial dysfunction plays a role in the development of sepsis-induced organ failure, their interaction and respective contribution to the disease remains poorly understood. Future research is necessary to better define such relationships in order to identify therapeutic targets and refine treatment strategies.

Topics: Capillary Permeability; Cardiovascular Agents; Cytochromes c; Erythropoietin; Humans; Microcirculation; Mitochondria; Mitochondrial Diseases; Poly Adenosine Diphosphate Ribose; Recombinant Proteins; Sepsis

Thirty years after Peter Mitchell was awarded the Nobel Prize for the chemiosmotic hypothesis, which links the mitochondrial membrane potential generated by the proton pumps of the electron transport chain to ATP production by ATP synthase, the molecular players involved once again attract attention. This is so because medical research increasingly recognizes mitochondrial dysfunction as a major factor in the pathology of numerous human diseases, including diabetes, cancer, neurodegenerative diseases, and ischemia reperfusion injury. We propose a model linking mitochondrial oxidative phosphorylation (OxPhos) to human disease, through a lack of energy, excessive free radical production, or a combination of both. We discuss the regulation of OxPhos by cell signaling pathways as a main regulatory mechanism in higher organisms, which in turn determines the magnitude of the mitochondrial membrane potential: if too low, ATP production cannot meet demand, and if too high, free radicals are produced. This model is presented in light of the recently emerging understanding of mechanisms that regulate mammalian cytochrome c oxidase and its substrate cytochrome c as representative enzymes for the entire OxPhos system.

Topics: Allosteric Regulation; Cytochromes c; Electron Transport Complex IV; Free Radicals; Humans; Membrane Potential, Mitochondrial; Mitochondrial Diseases; Models, Biological; Models, Molecular; Nitric Oxide; Oxidative Phosphorylation; Proton-Motive Force; Signal Transduction

Secondary injury following traumatic brain injury (TBI) is characterized by a variety of pathophysiologic cascades. Many of these cascades can have significant detrimental effects on cerebral mitochondria. These include exposure of neurons to excitotoxic levels of excitatory neurotransmitters with intracellular calcium influx, generation of reactive oxygen species, and production of peptides that participate in apoptotic cell death. Both experimental and clinical TBI studies have documented mitochondrial dysfunction, and animal studies suggest this dysfunction begins early and may persist for days following injury. Furthermore, interventions targeting mitochondrial mechanisms have shown neuroprotection after TBI. Continued evaluation and understanding of mitochondrial mechanisms contributing to neuronal cell death and survival after TBI is indicated. In addition, important underlying factors, such as brain maturation, that influence mitochondrial function should be studied. The ability to identify, target, and manipulate mitochondrial dysfunction may lead to the development of novel therapies for the treatment of adult and pediatric TBI.

Topics: Aging; Animals; Animals, Newborn; Apoptosis; Brain Injuries; Calcium; Cytochromes c; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Neurons; Neuroprotective Agents

Topics: Carnitine; Coenzymes; Cytochrome c Group; Cytochromes c; Dichloroacetic Acid; Drug Combinations; Drug Therapy, Combination; Flavin Mononucleotide; Humans; Mitochondrial Diseases; Succinic Acid; Thiamine; Thiamine Pyrophosphate; Ubiquinone

Hypoergos-energy deficiency, i.e., a mismatch between the body's (tissue, organ, cell) need for energy and the limited amount of macroergs (ATP) that can currently be used to maintain the structural integrity and functional activity of a tissue or organ. The main role in the development of hypoergoses is played by damage to mitochondria, therefore, in recent years, this term has been replaced by mitochondrial dysfunction. Over the past 50 years, drugs have been actively studied and developed that have the ability to influence mitochondrial disorders, both primary and secondary mitochondrial diseases. In this context, Cytochrome C, an original antioxidant/antihypoxant with a dual mechanism of action, deserves attention.. Энергетическая недостаточность — несоответствие между потребностью организма в энергии и тем ограниченным количеством макроэргов (АТФ), которое может в данный момент использоваться для поддержания его структурной целостности и функциональной активности ткани. Главную роль в развитии энергетической недостаточности играет повреждение митохондрий, поэтому в последние годы этот термин заменен на митохондриальную дисфункцию. Последние 50 лет активно изучаются и разрабатываются лекарственные средства, способные оказывать воздействие как на первичные, так и на вторичные митохондриальные заболевания. В этом контексте заслуживает внимания препарат Цитохром С — оригинальный антиоксидант/антигипоксант.

Topics: Adenosine Triphosphate; Antioxidants; Cytochromes c; Energy Metabolism; Humans; Mitochondria; Mitochondrial Diseases; Oxidative Stress

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]

Topics: Adenosine Triphosphate; Animals; Animals, Genetically Modified; Biological Transport; Cell Line; Cytochromes c; Electron Transport; Electron Transport Complex IV; HEK293 Cells; Humans; Hypoxia; Intracellular Signaling Peptides and Proteins; Kinetics; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Oxidation-Reduction; Respiration; Zebrafish

To determine whether high glucose (HG) or diabetes alters mitochondrial morphology and promotes mitochondrial fragmentation in retinal vascular cells and thereby triggers apoptosis associated with diabetic retinopathy.. To assess whether diabetes promotes mitochondrial fragmentation and thereby triggers apoptosis, retinas from nondiabetic and diabetic rats were analyzed using electron microscopy (EM) and in parallel, wild-type, diabetic, and OPA1+/- mice were analyzed for optic atrophy gene 1 (OPA1) and cytochrome c levels using Western blot (WB) analysis. To assess the relationship between mitochondrial fragmentation and OPA1 levels, rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mmol/L) medium, HG (30 mmol/L) medium, or in N medium transfected with OPA1 siRNA for seven days. Cells were examined for OPA1 expression and cytochrome c release by WB. In parallel, cells were stained with MitoTracker Red and assessed for mitochondrial fragmentation in live cells using confocal microscopy.. EM images revealed significant mitochondrial fragmentation in vascular cells of retinal capillaries of diabetic rats compared with that of nondiabetic rats. WB analysis showed significant OPA1 downregulation concomitant with increased levels of proapoptotic cytochrome c levels in cells grown in HG and in cells transfected with OPA1 siRNA alone. Similarly, OPA1 level was significantly reduced in diabetic retinas compared with that of nondiabetic retinas. Interestingly, OPA1+/- animals exhibited elevated cytochrome c release similar to those of diabetic mice.. Findings indicate that diabetes promotes mitochondrial fragmentation in retinal vascular cells, which are driven, at least in part, by decreased OPA1 levels leading to apoptosis in diabetic retinopathy.

Topics: Animals; Apoptosis; Blotting, Western; Cells, Cultured; Cytochromes c; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelium, Vascular; Glucose; GTP Phosphohydrolases; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Mitochondrial Diseases; Rats; Rats, Sprague-Dawley; Retinal Vessels; RNA, Small Interfering; Transfection

Impairment in mitophagy contributes to the pathology of Parkinson's disease. This study investigated whether Phosphatase and tensin homologue (PTEN)-induced kinase 1 (PINK1)/parkin-mediated mitophagy is linked to the protective effect of carnosic acid (CA) from rosemary. Treatment of SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) disrupted the mitochondrial membrane potential, inhibited voltage-dependent anion channel 1 (VDAC1) protein, and induced cytosolic cytochrome c, but CA pretreatment reversed these findings. By immunofluorescence, CA pretreatment was shown to increase the co-localization of red fluorescence (parkin) and MitoTracker green FM fluorescence (mitochondria), indicating that CA promoted the translocation of parkin into mitochondria. Immunoprecipitation with VDAC1 antibody showed that 6-OHDA treatment decreased the interaction of ubiquitinated protein with VDAC1. However, CA pretreatment reversed this reduction in the interaction of ubiquitinated protein with VDAC1. Silencing of PINK1 and parkin by use of small interfering RNA (siRNA) attenuated the ability of CA to reverse 6-OHDA-inhibited autophagic vacuoles. Moreover, in PINK1 siRNA-transfected cells, CA no longer reversed these actions of 6-OHDA on the inhibition of mitophagy-related proteins (PINK1, parkin, VDAC1, and LC3-II) and anti-apoptotic Bcl-2 protein, as well as the induction of apoptotic-related proteins, and nuclear condensation. In conclusion, CA appears to counteract the neurotoxicity of 6-OHDA by activating PINK1/parkin-mediated mitophagy.

Topics: Abietanes; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Mitophagy; Neuroprotective Agents; Oxidopamine; Protein Kinases; RNA, Small Interfering; Ubiquitin-Protein Ligases; Voltage-Dependent Anion Channel 1

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia and is maternally transmitted. Syndromic MD is a subgroup of MD including diabetic microangiopathy and macroangiopathy, in addition to extrapancreatic disorder. MD is caused by genetic mutations and deletions affecting mitochondrial DNA. This mitochondrial damage initiates apoptosis. In this study, we hypothesized that functional polymorphisms in genes involved in apoptotic pathway could be associated with the development of apoptosis in MD disease and increased its risk. Detection of apoptosis was confirmed on muscle biopsies taken from MD patients using the TUNEL method and the Cytochrome c protein expression level. We genotyped then 11 published SNPs from intrinsic and extrinsic apoptotic pathway and assessed the signification of these polymorphisms in 43 MD patients and 100 healthy controls. We found 10 selected polymorphisms (p53 (rs1042522 and rs17878362), BCL2 (rs2279115), BAX (rs1805419), BAK1 (rs210132 and rs2227925), CASP3 (rs1405937), CASP7 (rs2227310), CASP8 (rs1045485) and CASP10 (rs13006529)) with a potential apoptosis effect in MD patients compared to control population. Specifically, SNPs involved in the intrinsic pathway (p53, BCL2, BAK1 and CASP3) presented the highest risk of apoptosis. Our result proved that apoptosis initiated by mtDNA mutations, can be emphasized by a functional apoptotic polymorphisms associated with high expression of cytochrome c protein and more myofibers with apoptosis in syndromic MD subgroup compared with non-syndromic MD subgroup.

Topics: Adult; Apoptosis; Case-Control Studies; Cytochromes c; Diabetes Mellitus; Female; Genome-Wide Association Study; Humans; Linkage Disequilibrium; Male; Mitochondrial Diseases; Polymorphism, Single Nucleotide

An 8-year-old male Japanese Shiba exhibited muscle wasting and a stiff gait. A low-amplitude myotonic discharge was recorded by needle electromyography (EMG). A histopathological examination on a tru-cut biopsy sample from the muscle revealed myofiber size variations. Internal nuclei and cytoplasmic vacuoles were observed in many fibers. A type 1 fiber predominance and many hybrid type fibers were observed immunohistochemically. On the basis of these EMG and histopathological findings, myotonic dystrophy (DM) was suspected as tentative diagnosis. The cytoplasm around the vacuoles was immunopositive for cytochrome c, tom 20, and SOD-1, suggesting that these vacuoles might occur within mitochondria. Collectively, these results indicate that a mitochondrial abnormality partly play the role on the pathogenesis of present case.

Topics: Animals; Biopsy, Needle; Cytochromes c; Dog Diseases; Dogs; Electromyography; Male; Mitochondrial Diseases; Muscle, Skeletal; Muscular Diseases; Myotonic Dystrophy; Superoxide Dismutase-1

Anoxia leads to a robust generation of reactive oxygen species/nitrogen species which can result in mitochondrial dysfunction and associated cell death in the cerebral cortex of neonates.. The present study investigated the pharmacological role of tempol in the treatment of rat neonatal cortical mitochondrial dysfunction induced insult progression (day-1 to day-7) and associated neurobehavioral alterations post-anoxia.. Rat pups of 30h age or postnatal day 2 (PND2) were randomly divided into 5 groups (n=5 per group): (1) Control; (2) Anoxia; (3) Anoxia+Tempol 75mg/kg; (4) Anoxia+Tempol 150mg/kg; and (5) Anoxia+Tempol 300mg/kg, and subjected to two episode of anoxia (10min each) at 24h of time interval in an enclosed chamber supplied with 100% N. Tempol significantly decreased nitric oxide (NO) formation and simultaneously improved superoxide dismutase (SOD) and catalase (CAT) activities. Further, we observed a significantly (P<0.05) improvement in mitochondrial respiration, complex enzyme activities, mitochondrial membrane potential (MMP) along with attenuation of transition pore opening (MPT) after treatment with tempol. Furthermore, tempol decreased expression of mitochondrial Bax, cytochrome-C, caspase-9 and caspase-3 while the increase in expression of cytoplasmic Bax, mitochondrial Bcl-2 on day-7 in cortical region indicating regulation of intrinsic pathway of apoptosis. Further, it improved anoxia-induced neurobehavioral outcome (hanging and reflex latencies).. Biochemical, molecular and behavioral studies suggest the role of tempol in preserving mitochondrial function and associated neurobehavioral outcomes after neonatal anoxia.

Topics: Animals; Animals, Newborn; Antioxidants; Apoptosis; Catalase; Cyclic N-Oxides; Cytochromes c; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Hypoxia; Membrane Potential, Mitochondrial; Mitochondrial Diseases; Muscle Strength; NADH Dehydrogenase; Nitric Oxide; Proto-Oncogene Proteins c-bcl-2; Rats; Reflex; Spin Labels; Succinate Dehydrogenase; Superoxide Dismutase

Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used two-dimensional difference gel electrophoresis analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patients' fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes.

Topics: Actin Cytoskeleton; Actins; Antimycin A; Apoptosis; Cell Line, Tumor; Cell Survival; Cytochromes c; Electron Transport; Fibroblasts; Gelsolin; HeLa Cells; Humans; Mitochondria; Mitochondrial Diseases; Mitochondrial Membranes; Two-Dimensional Difference Gel Electrophoresis; Voltage-Dependent Anion Channel 1

Mitochondria, main producers of reactive-oxygen species (ROS), were studied to examine their role in the pathogenesis of systemic lupus erythematosus (SLE). PBMCs and mitochondria were isolated from SLE patients and healthy volunteers for various parameters. Mitochondrial ROS, swelling, hyperpolarization and levels of cytochrome c, caspase3 in the cells were assessed by flow cytometry. ROS was significantly increased in SLE patients (SLE vs controls: 1.83 ± 1.03 vs 1.10 ± 0.35; p < 0.0001). Depolarized state of mitochondria was greater in patients (SLE vs controls: 7.10 ± 5.50% vs 2.5 ± 1.8%; p < 0.05). Mitochondria swelling was found to be significantly altered in patients (SLE vs controls: 112.65 ± 36.56 vs 60.49 ± 20.69; p < 0.001). Expression of cytochrome c and caspase 3 (SLE vs controls: 1.37 ± 0.37% vs 1.01 ± 0.03%; 1.57 ± 0.46% vs 1.06 ± 0.07%; p < 0.05) respectively was found to be significantly increased in SLE. Further, the enzymatic activity of mitochondrial complex was assessed in isolated mitochondria. A significant decrease in activity of Complex I (SLE vs controls: 11.79 ± 3.18 vs 15.10 ± 6.38 nmol NADH oxidized/min/mg protein, p < 0.05); Complex IV (SLE vs control: 9.41 ± 5.16 vs 13.56 ± 5.92 nmol cytochrome c oxidized/min/mg protein, p < 0.05) and Complex V (SLE vs controls: 4.85 ± 1.39 vs 6.17 ± 2.02 nmol ATP hydrolyzed/min/mg protein, p < 0.05) was found in SLE patients in comparison to healthy controls. However, Complex II did not show significant variation in either group (SLE vs controls: 42.2 ± 28.6 vs 61.71 ± 42.3 nmol succinate oxidized/min/mg protein; ns). The decrease in enzyme activities of mitochondrial Complexes I, IV and V on one hand and ROS, hyperpolarization and apoptosis on the other points toward a possible role of mitochondria in the pathogenesis of lupus.

Topics: Adult; Apoptosis; Case-Control Studies; Caspase 3; Cytochromes c; Electron Transport Chain Complex Proteins; Female; Humans; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Mitochondrial Swelling; Reactive Oxygen Species

CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the "mitochondrial contact site and cristae organizing system" (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release.

Topics: Alleles; Apoptosis; Cell Line; Cytochromes c; DNA Repair; DNA, Mitochondrial; Genome, Mitochondrial; HeLa Cells; Humans; Hydrogen Peroxide; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Mutation; Oxidative Stress; Real-Time Polymerase Chain Reaction

Diabetes is a major driver of cardiovascular disease, but the underlying mechanisms remain elusive. Prolyl-isomerase Pin1 recognizes specific peptide bonds and modulates function of proteins altering cellular homoeostasis. The present study investigates Pin1 role in diabetes-induced vascular disease.. In human aortic endothelial cells (HAECs) exposed to high glucose, up-regulation of Pin1-induced mitochondrial translocation of pro-oxidant adaptor p66(Shc) and subsequent organelle disruption. In this setting, Pin1 recognizes Ser-116 inhibitory phosphorylation of endothelial nitric oxide synthase (eNOS) leading to eNOS-caveolin-1 interaction and reduced NO availability. Pin1 also mediates hyperglycaemia-induced nuclear translocation of NF-κB p65, triggering VCAM-1, ICAM-1, and MCP-1 expression. Indeed, gene silencing of Pin1 in HAECs suppressed p66(Shc)-dependent ROS production, restored NO release and blunted NF-kB p65 nuclear translocation. Consistently, diabetic Pin1(-/-) mice were protected against mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Increased expression and activity of Pin1 were also found in peripheral blood monocytes isolated from diabetic patients when compared with age-matched healthy controls. Interestingly, enough, Pin1 up-regulation was associated with impaired flow-mediated dilation, increased urinary 8-iso-prostaglandin F2α and plasma levels of adhesion molecules.. Pin1 drives diabetic vascular disease by causing mitochondrial oxidative stress, eNOS dysregulation as well as NF-kB-induced inflammation. These findings provide molecular insights for novel mechanism-based therapeutic strategies in patients with diabetes.

Topics: Analysis of Variance; Animals; Aorta; Case-Control Studies; Cells, Cultured; Chemokine CCL2; Cytochromes c; Diabetic Angiopathies; Endothelial Cells; Endothelium, Vascular; Gene Knockdown Techniques; Glucose; Humans; Hyperglycemia; Intercellular Adhesion Molecule-1; Male; Mice, Inbred C57BL; Mitochondrial Diseases; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Peptidylprolyl Isomerase; Reactive Oxygen Species; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Up-Regulation; Vascular Cell Adhesion Molecule-1; Vasculitis

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mtDNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5-fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electrons from the pentose phosphate pathway transferred via a 3-fold up-regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.

Topics: Aging; Animals; Cytochromes b5; Cytochromes c; DNA, Mitochondrial; Leydig Cells; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Diseases; Reactive Oxygen Species; Spermatozoa; Testosterone

Muscle dysfunction in acute organophosphorus (OP) poisoning is a cause of death in human. The present study was conducted to identify the mechanism of action of OP in terms of muscle mitochondrial dysfunction. Electromyography (EMG) was conducted on rats exposed to the acute oral dose of malathion (400 mg/kg) that could inhibit acetylcholinesterase activity up to 70%. The function of mitochondrial respiratory chain and the rate of production of reactive oxygen species (ROS) from intact mitochondria were measured. The bioenergetic pathways were studied by measurement of adenosine triphosphate (ATP), lactate, and glycogen. To identify mitochondrial-dependent apoptotic pathways, the messenger RNA (mRNA) expression of bax and bcl-2, protein expression of caspase-9, mitochondrial cytochrome c release, and DNA damage were measured. The EMG confirmed muscle weakness. The reduction in activity of mitochondrial complexes and muscular glycogen with an elevation of lactate was in association with impairment of cellular respiration. The reduction in mitochondrial proapoptotic stimuli is indicative of autophagic process inducing cytoprotective effects in the early stage of stress. Downregulation of apoptotic signaling may be due to reduction in ATP and ROS, and genotoxic potential of malathion. The maintenance of mitochondrial integrity by means of artificial electron donors and increasing exogenous ATP might prevent toxicity of OPs.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 9; Cell Death; Cytochromes c; Deoxyguanosine; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex IV; Glycogen; Insecticides; Lactic Acid; Malathion; Mitochondria, Muscle; Mitochondrial Diseases; Muscle, Skeletal; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species

An emerging literature suggests that bisphenol A (BPA), a widespread endocrine disrupting chemical, when exposure occurs in early life, may increase the risk of metabolic syndrome. In this study, we investigated the hypothesis that perinatal exposure to BPA predisposed offspring to fatty liver disease: the hepatic manifestation of metabolic syndrome, and its possible mechanism. Pregnant Wistar rats were administered with BPA (40μg/kg/day) or vehicle during gestation and lactation. Liver histology, biochemical analysis, transcriptome, and mitochondrial function were examined in male offspring at postnatal 3, 15 and 26 weeks. At 3 weeks of age, abnormal liver morphology and function were not observed in the BPA-exposed offspring, but a decrease in mitochondrial respiratory complex (MRC) activity (I and III) and significant changes in gene expression involved in mitochondrial fatty acid metabolism were observed compared with controls. At 15 weeks, micro-vesicular steatosis in liver, up-regulated genes involved in lipogenesis pathways, increased ROS generation and Cytc release were observed in the BPA-exposed offspring. Then, extensive fatty accumulation in liver and elevated serum ALT were observed in BPA-exposed offspring at 26 weeks. In the longitudinal observation, hepatic mitochondrial function including MRC activity, ATP production, ROS generation and mitochondrial membrane potential were progressively worsened in the BPA-exposed offspring. Perinatal BPA exposure contributes to the development of hepatic steatosis in the offspring of rats, which may be mediated through impaired hepatic mitochondrial function and up-regulated hepatic lipid metabolism.

Topics: Adenosine Triphosphate; Animals; Benzhydryl Compounds; Blotting, Western; Body Weight; Cytochromes c; Cytosol; Fatty Acids, Nonesterified; Fatty Liver; Female; Hepatocytes; Lipids; Liver; Male; Membrane Potential, Mitochondrial; Mitochondrial Diseases; Phenols; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Wistar; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Triglycerides

The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.

Topics: Animals; Apoptotic Protease-Activating Factor 1; Blotting, Western; Carrier Proteins; Cell Survival; Cells, Cultured; Cytochromes c; Fluorescent Antibody Technique; HEK293 Cells; HeLa Cells; Humans; Immunoprecipitation; Mice; Mitochondrial Diseases; Neoplasms; Neurons; Proteolysis; RNA Interference; Transferases; Ubiquitination

Patients with diabetes mellitus, particularly those with cardiovascular complications, have increased risk of mortality when subject to anesthetics and surgery, compared with non-diabetic patients. Anesthetics may exert pressure on the cardiovascular system of diabetic patients, directly or by aggravating pre-existing cardiovascular complications. Advanced glycation end products (AGEs) are extremely accumulated in diabetes mellitus, and are confirmed to play an important role in the pathogenesis of diabetic microvascular and macrovascular complications. The purpose of the present study was to investigate the regulatory role of etomidate, which is widely used as intravenous general anesthetics, on the viability and apoptosis of human endothelial Eahy926 cells, in the presence of AGEs. The results demonstrated that etomidate and Glu-BSA (one type of AGE) synergistically reduced the human endothelial Eahy926 cell viability and induced cell apoptosis. In addition, western blot assay of apoptosis-associated molecules indicated that both agents synergistically upregulated the cytochrome c release, activated the apoptosis executor, caspase 3, and promoted the poly-ADP-ribose polymerase (PARP) lysis. Further results confirmed that the two agents synergistically promoted oxidative stress by decreasing mitochondrial respiratory chain complex IV and mitochondrial membrane potential (MMP), while upregulating reactive oxygen species (ROS) and mitochondrial superoxide. In conclusion, the results presented in this study offer a novel insight into the mechanisms of endothelial cell apoptosis in response to etomidate in the presence of AGEs. These results suggest that oxidative stress has important role in the synergistic promotion of apoptosis by etomidate and AGEs in endothelial Eahy926 cells.

Topics: Anesthetics, Intravenous; Apoptosis; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Diabetes Mellitus; Diabetic Angiopathies; Drug Synergism; Endothelial Cells; Etomidate; Glycation End Products, Advanced; Humans; Mitochondrial Diseases; Oxidative Stress; Poly(ADP-ribose) Polymerases

Neurotoxicity of amyloid β (Aβ) plays an important role in Alzheimer's disease (AD) pathogenesis. In this study, we researched the potential protective effects of resistin against Aβ neurotoxicity in mouse Neuro2a (N2a) cells transfected with the Swedish amyloid precursor protein (Sw-APP) mutant and Presenilin exon 9 deletion mutant (N2a/D9), which overproduced Aβ with abnormal intracellular Aβ accumulation. The results show increased levels of ROS, NO, protein carbonyls, and 4HNE in N2a/D9 cells, which were attenuated by resistin treatment in a dose dependent manner. We also found that resistin could improve mitochondrial function in N2a/D9 cells through increasing the level of ATP and mitochondrial membrane potential. MTT and LDH assay indicated that N2a/D9 cells show increased vulnerability to H2O2-induced insult, which could be ameliorated by resistin. Mechanically, we found that resistin prevented apoptosis signals through reducing the ratio of Bax/Bcl2, the level of cleaved caspase-3, and attenuating cytochrome C release. Finally, the results demonstrated that resistin did not change the production of Aβ1-40 and Aβ1-42 in N2a/D9 cells, which suggests that the protective effects of resistin are independent of APP metabolism. This raises the possibility of novel AD therapies using resistin.

Topics: Adenosine Triphosphate; Aldehydes; Amyloid beta-Peptides; Animals; Apoptosis; Blotting, Western; Cell Line; Cell Survival; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Hydrogen Peroxide; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Diseases; Nitric Oxide; Oxidative Stress; Presenilin-1; Protein Carbonylation; Reactive Oxygen Species; Resistin; Signal Transduction

Reduced glutathione (GSH) has been shown to improve pulmonary graft preservation. Mitochondrial dysfunction is regarded to be the motor of ischemia-reperfusion injury (IR) in solid organs. We have shown previously that IR induces pulmonary mitochondrial damage. This study elucidates the impact of GSH preconditioning on the integrity and function of pulmonary mitochondria in the setting of warm pulmonary IR.. Wistar rats were subjected to control, sham, and to two-study-group conditions (IR30/60 and GSH-IR30/60) receiving IR with or without GSH preconditioning. Rats were anesthetized and received mechanical ventilation. Pulmonary in situ clamping followed by reperfusion generated IR. Mitochondria were isolated from pulmonary tissue. Respiratory chain complexes activities (I-IV) were analyzed by polarography. Mitochondrial viability (Ca2+-induced swelling) and membrane integrity (citrate synthase assay) were determined. Subcellular-fractional cytochrome C-content (Cyt C) was quantified by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was analyzed by fluorescence-activated cell sorting (FACS) after energizing and uncoupling. Inflammatory activation was determined by myeloperoxidase activity (MPO), matrix-metalloproteinase 9 (MMP-9) activity by gel zymography.. Pulmonary IR significantly reduced mitochondrial viability in combination with ΔΨm hyper-polarization. GSH preconditioning improved mitochondrial viability and normalized ΔΨm. Cyt C was reduced after IR; GSH protected from Cyt C liberation. Respiratory chain complex activities (I, II, III) declined during IR; GSH protected complex II function. GSH also protected from MMP-9 and neutrophil sequestration (P>.05).. GSH preconditioning is effective to prevent mitochondrial death and improves complex II function during IR, but not mitochondrial membrane stability. GSH-mediated amelioration of ΔΨm hyper-polarization appears to be the key factor of mitochondrial protection.

Topics: Animals; Apoptosis; Calcium; Cytochromes c; Disease Models, Animal; Drug Evaluation, Preclinical; Electron Transport; Glutathione; Granulocytes; Ischemic Preconditioning; Lung; Matrix Metalloproteinase 9; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Oxygen Consumption; Pulmonary Edema; Rats; Rats, Wistar; Reperfusion Injury

The objectives of this study were to determine whether neutrophil depletion with anti-neutrophil serum (ANS) or preconditioning with the hydrogen sulfide (H(2)S) donor NaHS (NaHS-PC) 24 h prior to ischemia-reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium-activated, large conductance potassium (BK(Ca)) channels were involved in this protective effect. I/R was induced by 45-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in rats preconditioned with NaHS (NaHS-PC) or a BK(Ca) channel activator (NS-1619-PC) 24 h earlier or treated with ANS. Mitochondrial function was assessed by measuring mitochondrial membrane potential, mitochondrial dehydrogenase function, and cytochrome c release. Mucosal myeloperoxidase (MPO) and TNF-α levels were also determined, as measures of postischemic inflammation. BK(Ca) expression in intestinal mucosa was detected by immunohistochemistry and Western blotting. I/R induced mitochondrial dysfunction and increased tissue MPO and TNF-α levels. Although mitochondrial dysfunction was attenuated by NaHS-PC or NS-1619-PC, the postischemic increases in mucosal MPO and TNF-α levels were not. The protective effect of NaHS-PC or NS-1619-PC on postischemic mitochondrial function was abolished by coincident treatment with BK(Ca) channel inhibitors. ANS prevented the I/R-induced increase in tissue MPO levels and reversed mitochondrial dysfunction. These data indicate that neutrophils play an essential role in I/R-induced mucosal mitochondrial dysfunction. In addition, NaHS-PC prevents postischemic mitochondrial dysfunction (but not inflammation) by a BK(Ca) channel-dependent mechanism.

Topics: Animals; Benzimidazoles; Cytochromes c; Hydrogen Sulfide; Intestinal Diseases; Intestine, Small; Ischemic Preconditioning; Leukocyte Reduction Procedures; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Neutrophils; Peroxidase; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sulfides; Tumor Necrosis Factor-alpha

Cisplatin nephropathy can be regarded as a mitochondrial disease. Intervention to halt such deleterious injury is under investigation. Recently, the flavanol (-)-epicatechin emerges as a novel compound to protect the cardiovascular system, owing in part to mitochondrial protection. Here, we have hypothesized that epicatechin prevents the progression of cisplatin-induced kidney injury by protecting mitochondria. Epicatechin was administered 8 h after cisplatin injury was induced in the mouse kidney. Cisplatin significantly induced renal dysfunction and tubular injury along with an increase in oxidative stress. Mitochondrial damages were also evident as a decrease in loss of mitochondrial mass with a reduction in the oxidative phosphorylation complexes and low levels of MnSOD. The renal damages and mitochondrial injuries were significantly prevented by epicatechin treatment. Consistent with these observations, an in vitro study using cultured mouse proximal tubular cells demonstrated that cisplatin-induced mitochondrial injury, as revealed by a decrease in mitochondrial succinate dehydrogenase activity, an induction of cytochrome c release, mitochondrial fragmentation, and a reduction in complex IV protein, was prevented by epicatechin. Such a protective effect of epicatechin might be attributed to decreased oxidative stress and reduced ERK activity. Finally, we confirmed that epicatechin did not perturb the anticancer effect of cisplatin in HeLa cells. In conclusion, epicatechin exhibits protective effects due in part to its ability to prevent the progression of mitochondrial injury in mouse cisplatin nephropathy. Epicatechin may be a novel option to treat renal disorders associated with mitochondrial dysfunction.

Topics: Animals; Antioxidants; Catechin; Cells, Cultured; Cisplatin; Cytochromes c; Disease Models, Animal; HeLa Cells; Humans; In Vitro Techniques; Kidney Diseases; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Diseases; Oxidative Stress; Reactive Oxygen Species; Succinate Dehydrogenase

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H(2)O(2)) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H(2)O(2) showed increases in miR-1 expression, mitochondria dysfunction, cytochrome-c release and apoptosis, Addition of IGF-1 into the iPS cell cultures reduced the H(2)O(2) cytotoxicity. Prediction algorithms showed that 3'-untranslated regions of IGF-1 gene as a target of miR-1. Moreover, miR-1 mimic, but not miR-1 mimic negative control, diminished the protective effect of IGF-1 on H(2)O(2)-induced mitochondrial dysfunction, cytochrome-c release and apoptosis in iPS cells. In conclusion, IGF-1 inhibits H(2)O(2)-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. IGF-1's effect is, at least partially, regulated by miR-1 in iPS cells.

Topics: 3' Untranslated Regions; Apoptosis; Cells, Cultured; Cytochromes c; Humans; Hydrogen Peroxide; Induced Pluripotent Stem Cells; Insulin-Like Growth Factor I; MicroRNAs; Mitochondria; Mitochondrial Diseases; Oxidative Stress

Massive hepatectomy (MHX) leads to failure of remnant livers. Excessive metabolic burden in remnant livers may cause mitochondrial dysfunction. This study investigated whether blockade of the mitochondrial permeability transition (MPT) with N-methyl-4-isoleucine cyclosporine (NIM811) improves the outcome of MHX.. Mice were gavaged with NIM811 (10 mg/kg before surgery and 5 mg/kg daily afterward) and underwent sham-operation or approximately 90% partial hepatectomy.. Serum alanine aminotransferase, necrosis, and apoptosis increased, respectively, to approximately 1200 U/L, 6.1%, and 7% after MHX. NIM811 decreased peak alanine aminotransferase release, necrosis, and apoptosis by 70%, 100%, and 42%, respectively. 5-Bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen expression, and the remnant liver weights were all increased significantly by NIM811 treatment, indicating improved liver regeneration. NIM811 also blunted hyperbilirubinemia by 54%, increased serum albumin by 51%, and improved survival from 6% to 40% after MHX. Hepatic mitochondrial depolarization, cell death, and MPT were detected by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. Mitochondrial depolarization occurred in many viable hepatocytes (13 cells/high-power field), and nonviable hepatocytes increased slightly to approximately 1 cell/high-power field at 3 hr after MHX. Entry of calcein into mitochondria after MHX indicated MPT onset. Importantly, NIM811 decreased mitochondria depolarization by more than 60%, blocked MPT onset, and prevented cell death. Decreases of hepatic ATP, mitochondrial cytochrome c release, and caspase-3 activation after MHX were also partially blocked by NIM811.. NIM811 minimized liver injury and improved liver regeneration after MHX, at least in part, by preventing MPT onset and subsequent compromised energy supply and proapoptotic cytochrome c release.

Topics: Adenosine Triphosphate; Alanine Transaminase; Animals; Caspase 3; Cell Death; Cyclosporine; Cytochromes c; Fluoresceins; Hepatectomy; Liver; Liver Regeneration; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondrial Diseases; Organ Size; Proliferating Cell Nuclear Antigen

Aplastic anaemia (AA) is a disease characterised by bone marrow hypocellularity and peripheral blood pancytopenia. AA is also associated with mitochondrial aberrations. The present study was undertaken primarily to test the hypothesis that a nutrient mixture could affect the nutritional rehabilitation of mitochondrial aberrations in AA mice. BALB/c AA mice were induced by a combination of hypodermic injections of acetylphenylhydrazine (100 mg/kg), X-rays (2·0 Gy) and intraperitoneal injections of cyclophosphamide (80 mg/kg). We treated these mice with nutrient mixture-supplemented diets in a dose-dependent manner (1445·55, 963·7, 674·59 mg/kg per d), and the effects of the nutrient mixture for mitochondrial rehabilitation were analysed in AA mice. Transmission electron microscopy showed that mitochondrial ultrastructural abnormalities in bone marrow cells, splenocytes and hepatocytes of the nutrient mixture groups were restored markedly, compared with the AA group. Mitochondrial membrane potentials of the nutrient mixture groups were increased remarkably. Western blot analysis also revealed that the nutrient mixture significantly inhibited cytochrome c release of mitochondria in the AA group. Furthermore, the mitochondrial DNA content of the nutrient mixture groups was also increased. Our data suggest that the nutrient mixture may promote the rehabilitation of mitochondrial aberrations, and consequently protects against mitochondrial dysfunction in AA mice.

Topics: Anemia, Aplastic; Animals; Blood Cell Count; Bone Marrow Cells; Cells, Cultured; Cytochromes c; Cytosol; Disease Models, Animal; DNA, Mitochondrial; Hemoglobins; Hepatocytes; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; Mitochondria; Mitochondrial Diseases; Nutritional Support; Random Allocation; Spleen

Benzo(a)pyrene (BaP) has been shown to be an inducer of apoptosis. However, mechanisms involved in BaP-induced mitochondrial dysfunction are not well-known. In this study, human fetal lung fibroblasts cells were treated with BaP (8, 16, 32, 64 and 128 μM) for 4 and 12 h. Cell viability, intracellular level of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), mitochondrial membrane potential (ΔΨ(m)) and cytochrome c release were determined. Changes in transcriptional levels of p53-dependent apoptotic genes (p53, APAF1, CASPASE3, CASPASE9, NOXA and PUMA) were measured. At time point of 4 h, BaP induced the intracellular ROS generation in 64 (p < .05) and 128 μM BaP groups (p < .01) but decreased the T-AOC activities in 32, 64 (p < .05 for both) and 128 μM BaP groups (p < .01). At time point of 12 h, ΔΨ(m) significantly decreased in ≥32 μM BaP groups (p < .05 for all). Amount of mitochondrial cytochrome c significantly increased in 128 μM BaP group (p < .01). Transcriptional levels of CASPASE3, CASPASE9, APAF1 and PUMA were up-regulated in all BaP groups (p < .05 for all) and in ≥32 μM groups for NOXA (p < .05). But only in 16 μM BaP group a relatively little expression of p53 mRNA was observed (p < .05). The results indicate that in the earlier period BaP promoted the generation of excessive ROS and subsequently the mitochondrial depolarization, whereas transactivations of the p53-dependent apoptotic genes were significantly induced at the later period.

Topics: Apoptosis; Benzo(a)pyrene; Carcinogens; Cell Line; Cell Survival; Cytochromes c; Fetus; Fibroblasts; Gene Expression; Humans; Lung; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Oxidative Stress; Reactive Oxygen Species; Ribosomal Protein S9; Ribosomal Proteins; Transcriptional Activation; Tumor Suppressor Protein p53; Up-Regulation

To investigate the antioxidative ability of a novel mitochondria-targeted peptide MTP-131 in immortalized human trabecular meshwork (iHTM) and glaucomatous human trabecular meshwork (GTM(3)) cell lines.. Cultured iHTM and GTM(3) cells were pretreated with MTP-131 for 1 hour, and sustained oxidative stress was induced by subjecting TM cells to 200 μM hydrogen peroxide (H(2)O(2)) for 24 hours. Untreated cells and cells incubated with H(2)O(2) alone were used as controls. Lactate dehydrogenase (LDH) assay was used to determine cell viability. Changes of mitochondrial membrane potential (ΔΨm) and generation of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry and confocal microscopy. Activation of caspase 3 was quantified by Western blotting, and apoptosis was measured by flow cytometry. Release of cytochrome c and changes in cytoskeleton were analyzed by confocal microscopy. Data were analyzed with commercial data analysis software and P < 0.05 was considered to be statistically significant.. In both iHTM and GTM(3) cells, decrease of ΔΨm and elevation of intracellular ROS were detected after sustained oxidative stress induced by H(2)O(2). When cells were pretreated with MTP-131, the H(2)O(2)-induced mitochondrial depolarization was prevented; intracellular ROS, LDH release, and apoptosis were significantly decreased; release of cytochrome c from mitochondria to cytoplasm and activation of caspase 3 were inhibited. In addition, cytoskeleton changes caused by H(2)O(2) were also alleviated by MTP-131.. Mitochondria-targeted peptide MTP-131 could prevent both iHTM and GTM(3) cells from sustained oxidative stress induced by H(2)O(2).

Topics: Annexin A5; Apoptosis; Blotting, Western; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Flow Cytometry; Humans; Hydrogen Peroxide; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Microscopy, Confocal; Mitochondria; Mitochondrial Diseases; Oligopeptides; Oxidative Stress; Reactive Oxygen Species; Trabecular Meshwork

The function of holocytochrome c synthase (HCCS, also called heme lyase) is to attach covalently the heme cofactor to cytochromes c in the mitochondria of animals, fungi and protozoa. Little is known about how the protein functions but CP motifs, commonly found in heme-binding proteins, are present. Here we examine holocytochrome c production by Saccharomyces cerevisiae HCCS in the Escherichia coli cytoplasm with emphasis on the conserved CP motifs long implicated in heme transfer by this enzyme. Unexpectedly, the two motifs, both towards the N-terminus, were not required for activity. Mutations in HCCS on the C-terminal side of the CP motifs, known to cause disease states in humans (microphthalmia with linear skin defects) abolished or drastically attenuated holocytochrome c production.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Conserved Sequence; Cytochromes c; Heme; Humans; Lyases; Mice; Mitochondria; Mitochondrial Diseases; Molecular Sequence Data; Mutation; Protein Binding; Saccharomyces cerevisiae

This work was designed to further study the mechanism by which sulforaphane (SFN) exerts a renoprotective effect against cisplatin (CIS)-induced damage. It was evaluated whether SFN attenuates the CIS-induced mitochondrial alterations and the impairment in the activity of the cytoprotective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and γ glutamyl cysteine ligase (γGCL). Studies were performed in renal epithelial LLC-PK1 cells and in isolated renal mitochondria from CIS, SFN or CIS+SFN treated rats. SFN effectively prevented the CIS-induced increase in reactive oxygen species (ROS) production and the decrease in NQO1 and γGCL activities and in glutathione (GSH) content. The protective effect of SFN on ROS production and cell viability was prevented by buthionine sulfoximine (BSO), an inhibitor of γGCL, and by dicoumarol, an inhibitor of NQO1. SFN was also able to prevent the CIS-induced mitochondrial alterations both in LLC-PK1 cells (loss of membrane potential) and in isolated mitochondria (inhibition of mitochondrial calcium uptake, release of cytochrome c, and decrease in GSH content, aconitase activity, adenosine triphosphate (ATP) content and oxygen consumption). It is concluded that the protection exerted by SFN on mitochondrial alterations and NQO1 and γGCL enzymes may be involved in the renoprotection of SFN against CIS.

Topics: Adenosine Triphosphate; Animals; Anticarcinogenic Agents; Antineoplastic Agents; Calcium Signaling; Cell Death; Cell Survival; Cisplatin; Cytochromes c; Enzyme Inhibitors; Glutamate-Cysteine Ligase; Glutathione; Isothiocyanates; Kidney; LLC-PK1 Cells; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; NAD(P)H Dehydrogenase (Quinone); Oxygen Consumption; Rats; Sulfoxides; Swine; Thiocyanates

Garcinol, derived from Garcinia indica and other related species, has been found to modulate several cell signalling pathways involved in apoptosis and cancer development. Growth arrest and DNA damage-inducible gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors; it is expressed at low levels under normal conditions but strongly induced upon growth arrest, DNA damage, and endoplasmic reticulum (ER) stress. This study investigated the effect of garcinol on Hep3B cells, a human hepatocellular cancer cell line lacking functional p53, with the goal of elucidating the molecular mechanisms of p53-independent apoptosis in hepatocellular cancer. Overall, garcinol activated not only the death receptor and the mitochondrial apoptosis pathways but also the ER stress modulator GADD153. Garcinol treatment led to the accumulation of reactive oxygen species (ROS), increased GADD153 expression, and reduced mitochondrial membrane potential. An increase in the Bax/Bcl-2 ratio resulted in enhanced apoptosis. Caspase-8 and tBid (truncated Bid) expression also increased in a time-dependent manner. The enzymatic activities of caspase-3 and caspase-9 increased approximately 13-fold and 7.8-fold, respectively. In addition, the proteolytic cleavage of poly-(ADP-ribose)-polymerase (PARP) and DNA fragmentation factor-45 (DFF-45) increased in dose- and time-dependent manners. Our data suggest a promising therapeutic application of garcinol in p53-independent apoptosis in cancers.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Arabidopsis Proteins; Breast Neoplasms; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Female; Hep G2 Cells; Humans; Intramolecular Transferases; Liver Neoplasms; Mitochondrial Diseases; Plant Extracts; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Reactive Oxygen Species; Terpenes; Transcription Factor CHOP

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disorder. Mutations in Cu,Zn superoxide dismutase (SOD1) cause approximately 20% of familial ALS. One of the possible mechanisms whereby they induce disease is mitochondrial dysfunction in motor neurons. Here we describe a patient with ALS and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation. Direct sequencing of SOD1 gene revealed a heterozygous mutation in codon 22 substituting a highly conserved amino acid, from glutamine to arginine (Q22R). Muscle biopsy showed a neurogenic pattern associated with cytochrome c oxidase (COX) deficiency in several muscle fibers. Western blot analysis demonstrated a reduction in SOD1 content in the cytoplasmic and mitochondrial fractions. These results suggest that a minute quantity of mutant SOD1 protein contributes to a mitochondrial toxicity also in muscle tissue.

Topics: Adult; Amyotrophic Lateral Sclerosis; Arginine; Cytochromes c; DNA Mutational Analysis; Family Health; Genetic Linkage; Genetic Predisposition to Disease; Glutamic Acid; Humans; Male; Mitochondria, Muscle; Mitochondrial Diseases; Mutation; Superoxide Dismutase; Superoxide Dismutase-1

OXPHOS deficits are associated with most reported cases of inherited, degenerative and acquired mitochondrial disease. Traditional methods of measuring OXPHOS activities in patients provide valuable clinical information but require fifty to hundreds of milligrams of biopsy tissue samples in order to isolate mitochondria for analysis. We have worked to develop assays that require less sample and here report novel immunocapture assays (lateral flow dipstick immunoassays) to determine the activities of complexes I and IV, which are far and away the most commonly affected complexes in the class of OXPHOS diseases. These assays are extremely simple to perform, rapid (1-1.5 h) and reproducible with low intra-assay and inter-assay coefficients of variability (CVs) s (<10%). Importantly, there is no need to purify mitochondria as crude extracts of whole cells or tissues are suitable samples. Therefore, the assays allow use of samples obtained non-invasively such as cheek swabs and whole blood, which are not amenable to traditional mitochondrial purification and OXPHOS enzyme analysis. As a first step to assess clinical utility of these novel assays, they were used to screen a panel of cultured fibroblasts derived from patients with isolated deficiencies in complex I or IV caused by identified genetic defects. All patients (5/5) with isolated complex IV deficiencies were identified in this population. Similarly, almost all (22/24) patients with isolated complex I deficiencies were identified. We believe that this assay approach should find widespread utility in initial screening of patients suspected of having mitochondrial disease.

Topics: Amino Acid Substitution; Biopsy; Cytochromes c; Electron Transport Complex I; Electron Transport Complex IV; Humans; Mitochondrial Diseases; Mutation; Oxidative Phosphorylation; Reproducibility of Results

The pathogenesis of mitochondrial disorders has largely focused on the impairment of cellular energy metabolism. However, mitochondrial dysfunction has also been implicated as a factor in the initiation of apoptosis due to the translocation of cytochrome c, from mitochondria to the cytosol, and the subsequent cleavage of pro-caspase 3. In this study, we determined the cytochrome c content of cytosols (skeletal muscle) prepared from 22 patients with evidence of compromised mitochondrial electron transport chain enzyme activity and 26 disease controls. The cytochrome c content of the mitochondrial electron transport chain-deficient group was found to be significantly (p < 0.02) elevated when compared with the control group (63.7 +/- 15.5 versus 27.7 +/- 2.5 ng/mg protein). Furthermore, a relationship between the cytosolic cytochrome c content of skeletal muscle and complex I and complex IV activities was demonstrated. Such data raise the possibility that mitochondrial cytochrome c release may be a feature of mitochondrial disorders, particularly for those patients with marked deficiencies of respiratory chain enzymes. Whether initiation of apoptosis occurs as a direct consequence of this cytochrome c release has not been fully evaluated here. However, for one patient with the greatest documented cytosolic cytochrome c content, caspase 3 could be demonstrated in the cytosolic preparation. Further work is required in order to establish whether a relationship also exists between caspase 3 formation and the magnitude of respiratory chain deficiency.

Topics: Adolescent; Adult; Caspase 3; Child; Child, Preschool; Citrate (si)-Synthase; Cytochromes c; Cytosol; Electron Transport; Humans; Indicators and Reagents; Infant; Infant, Newborn; Middle Aged; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Muscle, Skeletal; Young Adult

Nano-materials are currently being used in a variety of fields. One of the concerns associated with their use is their potential to harm human health. In an attempt to identify genes expressed differently in human lung cells (WI-26 VA4) exposed to nanosized (45 nm in diameter) PAMAM (polyamidoamine) dendrimers, we observed down-regulation of mitochondrial DNA-encoded genes involved in the maintenance of mitochondrial membrane potential. Down-regulation of gene expression was confirmed by semi-quantitative RT-PCR. Dendrimers were shown to colocalize with mitochondria and cause the release of cytochrome C. Mitochondrial membrane potential was disrupted and the viability of cells was decreased in the presence of dendrimers. Activation of caspases 3 and 9 was increased. Apoptosis was observed by annexin V/propidium iodide staining and DNA fragmentation. In summary, nanosized dendrimers damaged mitochondria resulting in apoptosis.

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Coloring Agents; Cytochromes c; Dendrimers; Enzyme Activation; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondrial Diseases; Mitochondrial Membranes; Nanoparticles; Propidium; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction

Alpha-synuclein is one of the main constituents of Lewy bodies and plays an important role in the pathology of Parkinson's disease. Mutation or overexpression of alpha-synuclein causes Parkinson's disease, and downregulation of alpha-synuclein resists MPP(+)-induced cell death, but the mechanism remains elusive. In this study, we attempted to explore the effect of alpha-synuclein knockdown on mitochondrial function in MPP(+)-treated SH-SY5Y cells. We reconstructed the short hairpin RNA expression vector, pGenesil-2, specially targeting alpha-synuclein mRNA, and it was stably transfected into SH-SY5Y cells. Cell viability, nuclear morphology, and mitochondrial membrane potential were then detected, and the expression of alpha-synuclein, cytochrome c, Bcl-2 and Bax were analyzed by Western blotting. The results showed that after exposure to 500 microM MPP(+) for 24 h, about 41.0+/-1.5% control cells showed low mitochondrial membrane potential. However, the percentage was 13.6+/-1.2% in MPP(+) treated alpha-synuclein knockdown cells. MPP(+) induced cytochrome c release significantly, which was about 3.1-fold compared with that of control. However, in alpha-synuclein knockdown cells, the release of cytochrome c was blocked, which was about 1.4-fold compared with that of control. The Bcl-2/Bax ratio of SH-SY5Y cells reduced to 35.5+/-3.8% after MPP(+) treatment, and this ratio was 85.2+/-3.0% in MPP(+) treated alpha-synuclein knockdown cells. These data suggest that knockdown of alpha- synuclein might be an effective means in rescuing MPP(+)-induced mitochondrial dysfunction of SH-SY5Y cells.

Topics: 1-Methyl-4-phenylpyridinium; alpha-Synuclein; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Nucleus Shape; Cell Survival; Central Nervous System Agents; Cytochromes c; Gene Knockdown Techniques; Humans; Inverted Repeat Sequences; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Neurons; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

To determine photoreceptor oxidative stress and damage in sympathetic ophthalmia (SO).. Immunohistologic study.. Eight formalin-fixed and paraffin-embedded human globes with typical histologic features of SO and five age-matched globes without intraocular inflammation (controls) were retrieved from the Doheny Eye Institute ophthalmic pathology files. Deparaffinized sections of the globes were processed to localize tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor-1 (TNF-R1), acrolein, inducible nitric oxide synthase (iNOS), and nitrotyrosine by immunolocalization method. The latter two were localized to photoreceptor mitochondria using anti-cytochrome C antibody. Apoptotic cells were detected by Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) assay and were localized to the site of oxidative stress using antinitrotyrosine antibody.. Increased expression of TNF-alpha can be seen in the photoreceptor nuclear layer in all SO globes, whereas no such expression was observed in control globes. TNF-R1, iNOS, acrolein, and nitrotyrosine were immunolocalized to the inner segments of the photoreceptors in all SO globes, but only mild focal staining was observed in the control retinas. Both nitrotyrosine and iNOS immunolocalization revealed positive staining restricted primarily to mitochondria at the inner segments of the photoreceptors. Most of the TUNEL-positive cells were detected in the photoreceptors at the site of nitrotyrosine staining. In contrast, the age-matched control globes showed negative results.. In SO, photoreceptor mitochondrial oxidative stress occurs in the absence of leukocytic infiltration of the retina and may lead to photoreceptor apoptosis and subsequent vision loss. The oxidative stress seems to be mediated by iNOS and TNF-alpha. The current anti-inflammatory therapy combined with agents that could prevent oxidative stress may prevent photoreceptor damage in SO and may preserve vision.

Topics: Acrolein; Adult; Aged; Apoptosis; Biomarkers; Cytochromes c; Fluorescent Antibody Technique, Indirect; Humans; In Situ Nick-End Labeling; Middle Aged; Mitochondrial Diseases; Nitric Oxide Synthase Type II; Ophthalmia, Sympathetic; Oxidative Stress; Photoreceptor Cells, Vertebrate; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha; Tyrosine

Oxidative phosphorylation (OXPHOS) deficiency results in a number of human diseases, affecting at least one in 5000 of the general population. Altering the function of genes by mutations are central to our understanding their function. Prior to the development of gene targeting, this approach was limited to rare spontaneous mutations that resulted in a phenotype. Since its discovery, targeted mutagenesis of the mouse germline has proved to be a powerful approach to understand the in vivo function of genes. Gene targeting has yielded remarkable understanding of the role of several gene products in the OXPHOS system. We provide a "tool box" of mouse models with OXPHOS defects that could be used to answer diverse scientific questions.

Topics: Adenine Nucleotide Translocator 1; Alkyl and Aryl Transferases; Animals; Apoptosis Inducing Factor; Cell Cycle Proteins; Cytochromes c; Disease Models, Animal; DNA Helicases; DNA-Binding Proteins; DNA, Mitochondrial; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Electron Transport Complex IV; High Mobility Group Proteins; Iron-Sulfur Proteins; Membrane Proteins; Mice; Mice, Knockout; Mitochondrial Diseases; Mitochondrial Proteins; Models, Animal; Oxidative Phosphorylation; Ribonucleotide Reductases; Superoxide Dismutase; Thymidine Kinase; Transcription Factors

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Topics: Animals; Cytochromes c; Electron Transport; Electron Transport Complex III; Electron Transport Complex IV; Male; Methacrylates; Mitochondria, Liver; Mitochondria, Muscle; Mitochondrial Diseases; Oxygen Consumption; Potassium Cyanide; Rats; Rats, Wistar; Thiazoles; Ubiquinone

The present study investigated the promoting effect of oxysterol 7-ketocholesterol against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 7-Ketocholesterol significantly enhanced the MPP(+)-induced nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, ascorbate, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of MPP(+) in the presence of 7-ketocholesterol. The results indicate that 7-ketocholesterol shows a synergistic effect against the cytotoxic effect of MPP(+). 7-Ketocholesterol may enhance the MPP(+)-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of reactive oxygen species and depletion of GSH. The findings suggest that 7-ketocholesterol as a promoting agent for the formation of mitochondrial permeability transition may enhance the toxic neuronal cell injury.

Topics: Animals; Caspase 3; Cell Death; Cell Differentiation; Cell Nucleus; Cell Survival; Cytochromes c; Dopamine Agents; Drug Synergism; Enzyme Inhibitors; Flow Cytometry; Glutathione; Ketocholesterols; Membrane Potentials; Mitochondria; Mitochondrial Diseases; MPTP Poisoning; PC12 Cells; Permeability; Rats; Reactive Oxygen Species

Topics: Animals; Calcium; Cytochromes c; Male; Membrane Potentials; Mitochondria; Mitochondrial Diseases; Molecular Structure; Rats; Rats, Wistar; Taurine

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.

Topics: Animals; Calcium; Calmodulin; Caspase 3; Caspases; Cell Count; Cell Death; Cell Size; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glutathione; Imidazoles; Leupeptins; Mitochondrial Diseases; PC12 Cells; Rats; Reactive Oxygen Species; Sulfonamides; Trifluoperazine

The goal of this investigation was to determine whether chenodeoxycholic acid (CDCA)-induced apoptosis is prevented by ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDC) and to characterize the involvement of mitochondria in the process. Cultured human HepG2 cells were treated in a dose- and time-dependent protocol in order to establish a sufficiently low exposure to CDCA that causes apoptosis but not necrosis. Low-dose CDCA induced an S-phase block and G2 arrest of the cell cycle, as determined by flow cytometry. As a result, cell proliferation was inhibited. CDCA-induced apoptosis, as determined by fluorescence microscopy of Hoechst 33342-stained nuclei, was evident upon coincubation with TUDC. Additionally, after exposure to UDCA plus CDCA, the cell membrane was permeable to fluorescent dyes. Caspase-9-like activity, poly(ADP-ribose) polymerase (PARP) cleavage, and extensive DNA fragmentation were detected in CDCA-exposed cells and in cells coincubated with TUDC, but not UDCA. CDCA caused a decrease in mitochondrial membrane potential and depletion of ATP, both of which were potentiated by UDCA but not TUDC. The results suggest that UDCA potentiates CDCA cytotoxicity, probably at the level of induction of the mitochondrial permeability transition (MPT). Consequently, as suggested by the lack of the main hallmarks of the apoptotic pathway, in the presence of UDCA, CDCA-induced apoptosis is not properly executed but degenerates into necrosis.

Topics: Adenosine Triphosphate; Apoptosis; Bile Acids and Salts; Bromodeoxyuridine; Caspase 9; Caspases; Cell Cycle; Cell Division; Cell Line; Cell Membrane Permeability; Cell Survival; Chenodeoxycholic Acid; Chromatin; Cytochromes c; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Humans; Mitochondrial Diseases; Necrosis; Poly(ADP-ribose) Polymerases; Taurochenodeoxycholic Acid; Time Factors; Tubulin; Ursodeoxycholic Acid

Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells.. Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay.. Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis.. Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.

Topics: Animals; bcl-2-Associated X Protein; Blotting, Western; Capillaries; Cell Death; Cell Membrane Permeability; Cytochromes c; Cytosol; Diabetes Mellitus, Experimental; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Male; Mitochondrial Diseases; Pericytes; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Retinal Diseases; Retinal Vessels

Cerebellar Purkinje neurons (PNs) are selectively vulnerable to AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepriopionic acid)-induced delayed neurotoxicity known as dark cell degeneration (DCD) that is expressed as cytoplasmic and nuclear condensation, neuron shrinkage, and failure of physiology. The present study was initiated to determine whether AMPA-receptor-induced DCD in PNs is associated with Bax translocation to the mitochondria, cytochrome C release from the mitochondria, changes in mitochondrial potential, and activation of representative initiator and executor caspases that include caspase-9, caspase-3, and caspase-7. AMPA consistently and rapidly hyperpolarized mitochondria as reflected by an increase in MitoTracker Red CMS Ros fluorescence. Increases in Bax immunoreactivity were quantitatively and temporally variable and Bax failed to localize to mitochondria. Additionally, we observed a marked increase in immunoreactivity of cytochrome C although its release from mitochondria was not apparent. Mitochondrial membrane hyperpolarization and increases in cytochrome C immunoreactivity preceded caspase activation. Immunohistochemical analyses revealed the active form of caspases-3 and -9 were markedly and significantly increased in PNs following 30 microM AMPA, and caspase-9 activation preceded caspase-3. Increases in active caspase-7 immunoreactivity were less frequently encountered in PNs. Thus DCD shares some characteristics of apoptotic programmed cell death, but lacks typical mitochondrial pathophysiology associated with classic apoptosis. These findings suggest that AMPA-induced DCD is a form of active PCD that lies on a spectrum between classical apoptosis and passive necrosis.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; bcl-2-Associated X Protein; Caspases; Cell Count; Cerebellum; Cytochromes c; Electron Transport Complex IV; Enzyme Activation; Fluorescent Dyes; Immunohistochemistry; In Vitro Techniques; Microscopy, Confocal; Mitochondrial Diseases; Nerve Degeneration; Organic Chemicals; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Purkinje Cells; Rats; Rats, Sprague-Dawley; Time Factors