ubiquinone and thiazolyl-blue

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.

Topics: Analysis of Variance; Animals; Astrocytes; Blotting, Western; Cell Respiration; Cell Survival; Dose-Response Relationship, Drug; Immunohistochemistry; Membrane Potential, Mitochondrial; Mice; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Time Factors; Ubiquinone; Ultraviolet Rays

Idebenone is a coenzyme Q10 analog and an antioxidant that has been used clinically to treat Friedreich Ataxia. Being an antioxidant, idebenone could have potential therapeutic potential to treat other neurodegenerative diseases such as Parkinson's disease in which oxidative stress plays a role in their pathogenesis. But whether idebenone can be used to treat Parkinson's disease has not been evaluated. In this study, we found that exposure of the dopaminergic neuroblastoma SHSY-5Y cells to 1-10 μM idebenone for 72 h had no effect on the cell viability revealed by trypan blue exclusion assay and MTT assay. However, cells exposed to 25 μM or higher concentrations of idebenone showed extensive trypan blue-positive staining and significant reduction in cell viability revealed by MTT assay indicating that most of the cells were no longer viable. Idebenone-induced cell death was characterized by genomic DNA fragmentation and accumulation of cytochrome c in the cytosol indicating that the death was apoptotic in nature. In addition, idebenone induced an increase in the total RNA of the pro-apoptosis protein BAX, it also increased the caspase-3 activity in the cell lysates when compared with the untreated control cells or cells exposed to 10 μM or lower concentrations of idebenone. The detrimental effect of idebenone was attenuated by glutathione, an antioxidant, suggesting that oxidative stress contributed to the idebenone-induced cell death. In conclusion, our results suggest that antioxidant idebenone induced apoptosis when used in high concentrations.

Topics: Antioxidants; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Glutathione; Humans; Neuroblastoma; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue; Ubiquinone

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF(3)), a phospholipase A(2) (PLA(2)) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.

Topics: Adenine; Allopurinol; Animals; Antioxidants; Apoptosis; Arachidonic Acids; Benzoquinones; Blotting, Western; Calcium; Carbon; Caspase 3; Caspases; Cell Death; Cell Survival; Chick Embryo; Chromans; Coloring Agents; Cytosol; DNA Fragmentation; Enzyme Inhibitors; Glutathione; In Situ Nick-End Labeling; Indazoles; Metalloporphyrins; Mitochondria; Neurons; Nitric Oxide Synthase; Oligomycins; Protein Isoforms; Reactive Oxygen Species; Retina; Rotenone; Staurosporine; Tetrazolium Salts; Thiazoles; Time Factors; Ubiquinone; Uncoupling Agents; Xanthine Oxidase

We studied the potential neuroprotective action of nicergoline in immortalized hypothalamic GT1-7 cells exposed to agents which deplete levels of reduced glutathione, thus causing oxidative stress and cell death. Treatment with diethylmaleate (1 mM), buthionine sulfoximine (500 microM) or menadione (10-50 microM) caused diffuse GT1-7 cell degeneration, as assessed by using either the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay or the fluorescent dyes fluorescein diacetate and propidium iodide. Pre- and/or co-exposure of the cells to nicergoline significantly prevented diethylmaleate- or buthionine sulfoximine-induced neuronal death, whereas nicergoline was ineffective against menadione-induced toxicity. This effect was concentration-dependent and was mimicked by the classical antioxidants idebenone and vitamin E, and did not depend on interference with protein kinase C. Interestingly, the antineurodegenerative activity of nicergoline and vitamin E or idebenone was not additive, suggesting that these compounds share some intracellular mechanism(s) responsible for their protective effects. In conclusion, the present data indicate that nicergoline has neuroprotective activity, possibly mediated by the antioxidant activity of the molecule, and give support to the potential use of nicergoline in the prevention and therapy of neurodegenerative diseases.

Topics: Animals; Antioxidants; Benzoquinones; Buthionine Sulfoximine; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Maleates; Neurons; Neuroprotective Agents; Nicergoline; Staurosporine; Tetrazolium Salts; Thiazoles; Ubiquinone; Vitamin E

The effect of energetic metabolism compromise, obtained by chemical induction of hypoglycaemia (glucose deprivation), hypoxia (mitochondrial respiratory chain inhibition), and ischaemia (hypoglycaemia plus hypoxia), on glutamate toxicity was analyzed on PC12 cells. The respiratory status of these cells, measured by the MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay, was significantly decreased after metabolic inhibition induced by ischaemia, but it was not affected by both hypoglycaemia and hypoxia. Under hypoglycaemia, but not under hypoxia, ATP levels were significantly reduced (from 12.67+/-0.48 to 5.38+/-1.41 nmol/mg protein). However, ischaemic-like conditions greatly potentiated the decline of ATP levels (95% decrease) observed after hypoglycaemia. The influence of metabolic inhibition on glutamate-induced cytotoxicity was also analyzed. When the cells were preincubated under conditions that deplete ATP (hypoglycaemia and ischaemia), the inhibition of MTT reduction, measured after glutamate incubation, was potentiated. This effect could be reverted when vitamin E and idebenone were present during the induction of metabolic inhibition. The ATP levels above which glutamate susceptibility was enhanced were also determined. These results indicate that glutamate toxicity on PC12 cells, which occurs by a mechanism independent of N-methyl-D-aspartate (NMDA) receptor activation, can be enhanced by the depletion of intracellular ATP upon metabolic stress; it is dependent on the extent of ATP depletion and seems to involve the generation of free radicals. It can be concluded that under ischaemic conditions, the deleterious effects of glutamate can be potentiated by the energetic compromise associated with this pathologic situation.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Benzoquinones; Cell Hypoxia; Disease Susceptibility; Energy Metabolism; Glutamic Acid; Hypoglycemia; Ischemia; Mitochondria; PC12 Cells; Rats; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Thiazoles; Ubiquinone; Vitamin E

Two different tetrazolium compounds were compared for use in a colorimetric assay for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Brucella abortus. The tetrazolium compounds tested included 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sodium 3,3'-[1[(phenylamino)carbonyl]-3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT). The MTT and XTT colorimetric bactericidal assays were conducted by incubating antibody-opsonized bacteria with neutrophils in microtiter plates for 30 and 60 min at ratios of ten and 100 bacteria per neutrophil. Neutrophils were then lysed with saponin and samples were incubated 30 min with MTT or XTT plus coenzyme Q (CQ). Dead bacteria and lysed neutrophils did not react with MTT or XTT plus CQ. Live bacteria converted XTT to water soluble orange formazan in the presence of CQ and MTT to insoluble purple formazan. Absorption of formazan produced by bacteria from XTT was measured at 450 nm. Formazan produced by bacteria from MTT was solubilized by adding isopropanol and measured by absorption at 560 nm. Absorption of both types of formazan was directly related to viable bacteria cell number and used to determine the number of bacteria not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT or XTT plus CQ with known numbers of bacteria. The XTT and MTT colorimetric bactericidal assays produced comparable results when used to measure bovine neutrophil bactericidal activity against S. aureus, E. coli, L. monocytogenes, and B. abortus. However, the assay using XTT was quicker and easier to perform because bacteria converted XTT to a formazan that did not need to be solubilized before measuring absorption.

Topics: Animals; Blood Bactericidal Activity; Cattle; Colorimetry; Neutrophils; Tetrazolium Salts; Thiazoles; Ubiquinone