cytochrome-c-t and 6-hydroxy-2-5-7-8-tetramethylchroman-2-carboxylic-acid

Redox imbalance due to hyperglycemia is a causative factor for an increased generation of reactive oxygen species (ROS) that leads to mitochondrial dysfunction and the release of cytochrome-c. The aim of the present study is to elucidate the functional role of oxidative stress (OS) in the induction of apoptosis in H9c2 cells in the hyperglycemic state through glucose transporter-4 (GLUT-4) regulation and antioxidant status. H9c2 cells were incubated with 15, 24, and 33 mM glucose for 24, 48, and 72 hr to induce hyperglycemic stress. Hyperglycemic episodes have significantly influenced GLUT-4 mRNA regulation, depleted glutathione (GSH) and its associated enzymes, reduced cellular antioxidant enzymes (AOEs), caused nuclear condensation, and induced apoptosis by activating caspase-9 and 3 and annexin V binding in a concentration and duration-dependent manner. Trolox pretreatment significantly enhanced the GLUT-4 mRNA and antioxidant defense mechanism, suppressed nuclear condensation, and prevented cytochrome-c release, thereby reducing mitochondrial-dependent apoptosis. The present study shows that the toxic effect of high glucose is significantly regulated and that OS induction can be prevented through a water-soluble vitamin E analog "Trolox" treatment.

Topics: Animals; Antioxidants; Apoptosis; Chromans; Cytochromes c; Enzymes; Glucose; Glucose Transporter Type 4; Glutathione; Hyperglycemia; Myocytes, Cardiac; Oxidative Stress; Rats

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

Topics: Algorithms; Animals; Antioxidants; Butylated Hydroxytoluene; Cardiolipins; Chromans; Cytochromes c; Dextrans; Fluoresceins; Hydrogen Peroxide; Lipid Peroxidation; Liposomes; Models, Chemical; Models, Molecular; Oxidants; Permeability; Protein Binding; Quercetin; Rhodamines; Spectrometry, Fluorescence

Acrylamide (ACR) can be produced during food processing and has neurotoxic effects in humans. This study aims to determine ACR induced apoptotic responses in human astrocytoma U-1240 MG cells to realize the incurred toxic mechanisms. Under 1 and 2mM ACR exposure, cell viability decreased as time increased. The increments in sub-G(1) phase were 87.5-fold, and pro-caspase 3 and PARP protein expressions decreased 35% and 54.5% respectively relative to the control after 2mM ACR treatment. Molecular evidence of Bax/bcl-2 ratio and cytochrome c expression increased 8.86-fold and 6.81-fold as well as pro-caspase 9 decreased 67.8% relative to the control respectively under 2mM ACR exposure. Trolox, an ROS scavenging agent, attenuated cell death and induced ROS production by 2mM ACR. The ultrastructure alterations of mitochondria showed marked vesicular matrix compartmentalization and cytoplasmic vacuole formation after 2mM ACR was treated for 48h, whereas those treated for 72h showed chromatin condensation, pyknosis, and swelling. These results indicate long-term exposure to ACR induced mitochondria collapse and finally led to apoptosis. Although 2mM ACR is higher than average daily intake dosage, workers in chemical industries may be exposed to sufficient doses to entail health risks.

Topics: Acrylamide; Apoptosis; Astrocytoma; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Chromans; Cytochromes c; Dose-Response Relationship, Drug; G1 Phase; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Toxicity Tests

T-2 toxin, a member of the trichothecene mycotoxin family produced by the Fusarium fungi, has been shown to exert a variety of toxic effects on multiple targets in vivo. However, the embryonic toxicity of T-2 toxin in vitro remains unclear. In the present study, two permanent cell lines, embryonic stem cells (ES cells D3) and fibroblast 3T3 cells, were used to evaluate T-2 toxin toxicity. Differentiated mouse ES cells were cultivated as embryoid bodies along with T-2 toxin at different concentrations (0.5, 1, and 2 ng/ml) for 24 h. The increases in cellular reactive oxygen species (ROS), lipid and DNA oxidative damage, and loss of mitochondrial transmembrane potential were observed at 1 and 2 ng/ml concentrations. Flow cytometry showed that T-2 toxin induced cell cycle arrest and apoptosis. Furthermore, T-2 toxin opened the mitochondrial permeability transition pore, caused the release of cytochrome c from mitochondria and induced the upregulation of p53, caspase-9, caspase-3 expression and increased the ratio of Bax/Bcl-2. However, T-2 toxin-induced oxidative damage and apoptosis in differentiated ES cells decreased significantly in the presence of the antioxidant Trolox. Taken together, these results demonstrate that T-2 toxin induces oxidative stress and apoptosis in differentiated murine ES cells, and ROS-mediated mitochondrial pathway plays an important role in T-2 toxin induced apoptosis.

Topics: 3T3 Cells; Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Cell Differentiation; Cell Survival; Chromans; Coculture Techniques; Cytochromes c; DNA Damage; Embryoid Bodies; Embryonic Stem Cells; G1 Phase Cell Cycle Checkpoints; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Reactive Oxygen Species; T-2 Toxin; Teratogens

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity at supraclinical concentrations via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies.

Topics: Anesthetics, Dissociative; Antioxidants; Apoptosis; Cell Differentiation; Chromans; Cytochromes c; Dose-Response Relationship, Drug; Embryonic Stem Cells; Humans; In Vitro Techniques; Ketamine; Membrane Potential, Mitochondrial; Mitochondria; Neurons; Neurotoxicity Syndromes; Oxidative Stress; Reactive Oxygen Species; Time Factors

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 microM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 microM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 microM.

Topics: Animals; Antioxidants; Apoptosis; Chromans; Cytochromes c; Dose-Response Relationship, Drug; Flavonoids; Free Radicals; Liver; Molecular Structure; Peroxidase; Quercetin; Rutin; Superoxides

Amiodarone (AM), a drug used in the treatment of cardiac dysrrhythmias, can produce severe pulmonary adverse effects, including fibrosis. Although the pathogenesis of AM-induced pulmonary toxicity (AIPT) is not clearly understood, several hypotheses have been advanced, including increased inflammatory mediator release, mitochondrial dysfunction, and free-radical formation. The hypothesis that AM induces formation of reactive oxygen species (ROS) was tested in an in vitro model relevant for AIPT. Human peripheral lung epithelial HPL1A cells, as surrogates for target cells in AIPT, were susceptible to the toxicity of AM and N-desethylamiodarone (DEA), a major AM metabolite. Longer incubations (> or =6 h) of HPL1A cells with 100 microM AM significantly increased ROS formation. In contrast, shorter incubations (2 h) of HPL1A cells with AM resulted in mitochondrial dysfunction and cytoplasmic cytochrome c translocation. Preexposure of HPL1A cells to ubiquinone and alpha-tocopherol was more effective than that with Trolox C or 5,5-dimethylpyrolidine N-oxide (DMPO) at preventing AM cytotoxicity. These data suggest that mitochondrial dysfunction, rather than ROS overproduction, represents an early event in AM-induced toxicity in peripheral lung epithelial cells that may be relevant for triggering AIPT, and antioxidants that target mitochondria may potentially have beneficial effects in AIPT.

Topics: alpha-Tocopherol; Amiodarone; Anti-Arrhythmia Agents; Cell Line; Chromans; Cyclic N-Oxides; Cytochromes c; Cytoplasm; Epithelial Cells; Humans; Lung; Mitochondria; Reactive Oxygen Species; Ubiquinone

Arsenic trioxide (As2O3) is an effective therapy in acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the higher concentrations required to induce apoptosis. We have reported that trolox, an analogue of alpha-tocopherol, increases As2O3-mediated apoptosis in a variety of APL, myeloma and breast cancer cell lines, while non-malignant cells may be protected. In the present study, we extended previous results to show that trolox increases As2O3-mediated apoptosis in the P388 lymphoma cell line in vitro, as evidenced by decrease of mitochondrial membrane potential and release of cytochrome c. We then sought to determine whether this combination can enhance antitumor effects while protecting normal cells in vivo. In BDF1 mice, trolox treatment decreased As2O3-induced hepatomegaly, markers of oxidative stress and hepatocellular damage. In P388 tumor-bearing mice, As2O3 treatment prolonged survival, and the addition of trolox provided a further significant increase in lifespan. In addition, the combination of As2O3 and trolox inhibited metastatic spread, and protected the tumor-bearing mice from As2O3 liver toxicity. Our results suggest, for the first time, that trolox might prevent some of the clinical manifestations of As2O3-related toxicity while increasing its pro-apoptotic capacity and clinical efficacy in hematological malignancies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Chromans; Cytochromes c; Drug Synergism; Humans; Liver; Lymphoma; Membrane Potential, Mitochondrial; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Oxides