vitamin-k-semiquinone-radical and thiazolyl-blue

Vitamin K2 [menaquinone-4 (MK-4)] has been reported to induce apoptosis in hepatocellular carcinoma, leukemia, and MDS cell lines. The effects of MK-4 on the development of arthritis have never been addressed so far. In this study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis (CIA). We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes (FLSs) using the 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The proapoptotic effect of MK-4 upon FLS was investigated with annexin V staining and DNA fragmentation and caspase 3/7 assays. Moreover, we analyzed the effect of MK-4 on the development of CIA in female dark agouti rats. Our results indicated that MK-4 inhibited the proliferation of FLS and the development of CIA in a dose-dependent manner. We concluded that MK-4 may represent a new agent for the treatment of RA in the setting of combination therapy with other disease-modifying antirheumatic drugs.

Topics: Animals; Antirheumatic Agents; Apoptosis; Arthritis, Rheumatoid; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Models, Biological; Rats; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Vitamin K

Linkage of alpha-synuclein (alpha-SN) mutations to familial Parkinson's disease (PD) and presence of alpha-SN as a major constituent of Lewy body in both sporadic and familial PD implicate alpha-SN abnormality in PD pathogenesis. Here we demonstrate that overexpression of wild-type or mutant alpha-SN does not cause any deleterious effect on the growth or continued propagation of transfected human cells, but overproduction of mutant alpha-SN heightens their sensitivity to menadione-induced oxidative injury. Such enhanced vulnerability is more pronounced in neuronal transfectants than in their nonneuronal counterparts and is associated with increased production of reactive oxygen species. The data suggest that mutated alpha-SN, especially with an alanine-to-proline substitution at residue 30, sensitizes neuronal cells to oxidative damage.

Topics: alpha-Synuclein; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Humans; Immunoblotting; Kidney; Mutation; Nerve Tissue Proteins; Neurons; Oxidative Stress; Parkinson Disease; Reactive Oxygen Species; RNA, Messenger; Sulfhydryl Compounds; Synucleins; Tetrazolium Salts; Thiazoles; Transfection; Vitamin K

Benzophenone is an ultraviolet (UV)-absorbing agent that has been used in industry and medicine for more than 30 years. Consumers of cosmetics and sunscreens containing UV-absorbers are exposed to benzophenones on a daily basis, owing to the widespread use of these compounds. However, the efficacy of these compounds as scavengers of oxidative stress is still not well established. In the present study, we investigate the antioxidative capacity of six sunscreen benzophenone compounds. A primary myoblast culture was mixed in vitro with 100 microM menadione. The cytotoxic effect by menadione-induced oxidative stress was monitored by the lucigenin- or luminol-amplified chemiluminescence, methylthiotetrazole (MTT) assay, and the antioxidative effects of various benzophenone compounds were evaluated. The results showed that the addition of menadione can induce oxidative stress on myoblasts by superoxide and hydrogen peroxide production, which can be eradicated by superoxide dismutase (SOD) and catalase, respectively, in a dose-dependent mode. The catalase has a protective effect on the cytotoxicity induced by menadione as measured by the MTT assay, while the SOD does not. The selected benzophenones also have a significant scavenging effect on the menadione-induced cell death on the myoblasts. The ortho-dihydroxyl structure and other hydroxy groups in the same ring have a stronger scavenging effect on the superoxide anion on myoblasts; thus, a stable penoxy radical may be formed. The mechanism of this effect remains to be clarified.

Topics: Acridines; Animals; Benzophenones; Cell Survival; Drug Evaluation, Preclinical; Free Radical Scavengers; Humans; In Vitro Techniques; Luminescent Measurements; Luminol; Muscle, Skeletal; Oxidative Stress; Rats; Sunscreening Agents; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Vitamin K

The toxicity of allyl alcohol, coumarin and menadione has been studied in precision-cut liver slice cultures. Liver slices were prepared from male Sprague-Dawley rats, male Dunkin-Hartley guinea-pigs and from samples of Cynomolgus monkey and human liver using a Krumdieck tissue slicer. The liver slices were cultured with the test compounds for 24 h in a dynamic organ culture system. Toxicity was assessed by measurement of protein synthesis, potassium content and the MTT assay. At the concentrations examined, menadione produced marked toxicity in liver slices from all four species, whereas rat liver slices were less susceptible to allyl alcohol toxicity. Coumarin produced concentration-dependent toxic effects in rat and guinea-pig liver slices, whereas Cynomolgus monkey and human liver slices were relatively resistant, especially at low coumarin concentrations. At some concentrations of the test compounds examined, the MTT assay appeared to be a less sensitive indicator of toxicity than either protein synthesis or potassium content. These results demonstrate the usefulness of precision-cut liver slices for assessing species differences in xenobiotic-induced toxicity.

Topics: 1-Propanol; Animals; Coloring Agents; Coumarins; Guinea Pigs; Humans; Liver; Macaca fascicularis; Male; Potassium; Propanols; Protein Biosynthesis; Rats; Tetrazolium Salts; Thiazoles; Toxicity Tests; Vitamin K

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30 degrees C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.

Topics: Amphotericin B; Aspergillus fumigatus; Candida albicans; Dose-Response Relationship, Drug; Fluconazole; Formazans; Itraconazole; Microbial Sensitivity Tests; Mitochondria; Tetrazolium Salts; Thiazoles; Vitamin K

A MTT/formazan assay was used to evaluate the antitumor activity of vitamin C (Vit C), vitamin K3 (Vit K3), or vitamin C:vitamin K3 combinations against a human prostatic carcinoma cell line (DU145). Both Vit C and Vit K3 alone exhibited antitumor activity, but only at elevated doses. When Vit C and Vit K3 were combined at a C:K3 ratio of 100:1 and administered to the carcinoma cells, the 50% cytotoxic concentrations (CD50) of the vitamins decreased 10- to 60-fold. Subsequently, the DU145 cells were examined with transmission and scanning electron microscopy (TEM and SEM) following a 1 hour treatment with Vit C, Vit K3, or Vit C/K3 combined at their 50% cytotoxic dose. Our morphological data suggest that vitamin treatment with individual vitamins affects the cytoskeleton, the mitochondria, and other membranous components of the cell. Treatment with the vitamin combination appears to potentiate the effects of the individual vitamin treatment. Specifically, there are abundant necrotic cells. The surviving cells display morphological defects characteristic of cell injury.

Topics: Antineoplastic Agents; Ascorbic Acid; Carcinoma; Cell Membrane; Coloring Agents; Cytoskeleton; Drug Synergism; Drug Therapy, Combination; Humans; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Mitochondria; Prostatic Neoplasms; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vitamin K

A modification of the MTT based tetrazolium colorimetric assay is described. Using the electron-coupling agent menadione formazan formation by murine splenocytes and P-815 cells was significantly increased whereas dye reduction by macrophages was hardly influenced. These observations suggest that it should be possible to improve the tetrazolium based cytotoxicity assays of murine macrophages against cells of the syngeneic tumour cell line P-815.

Topics: Animals; Colorimetry; Coloring Agents; Cytotoxicity Tests, Immunologic; Dose-Response Relationship, Drug; Macrophages; Mice; Mice, Inbred CBA; Oxidation-Reduction; Rats; Rats, Wistar; Spleen; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vitamin K

Vitamin K (VK) congeners (VK1, VK2, and VK3) have been used as antihemorrhagic agents, while VK3 has also been found to inhibit growth in various rodent and human tumor cells. We have compared the antitumor activities of vitamin K1, K2, and K3 against a panel of human cancer cell lines. For each test agent, a dose-response profile was generated by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and an SRB (sulforhodamine B) assay. Both assays yielded similar results. The respective ID50 values of VK3 in five hepatoma cell lines, HA59T, HA22T, PLC, HepG2, and Hep3B, of increasing differentiation state, were 42, 36, 28, 27, and 20 microM. For nasopharyngeal carcinoma (CG1), leukemia (U937), oral epidermoid carcinoma (KB), and breast carcinoma (BC-M1) cells, the ID50 values of VK3 were 26, 15, 25, and 33 microM, respectively. For all the above cells, the ID50 values of VK1 ranged from 6 to 9 mM, and the ID50 values of VK2 ranged from 1 to 2 mM. Thus, the relative potencies of antitumor activity of VK3 compared to VK2 and to VK1 are about 60- and 300-fold, respectively. These results support the preference for use of VK3 over VK1 and VK2 in cancer therapy.

Topics: Antineoplastic Agents; Cell Survival; Drug Screening Assays, Antitumor; Glutathione; Humans; Rhodamines; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vitamin K

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Antineoplastic Agents; Cell Count; Cell Line; Cell Survival; Coloring Agents; Humans; Hydrogen Peroxide; Indicators and Reagents; Luminescent Measurements; Male; Mice; Prostatic Neoplasms; Tetrazolium Salts; Thiazoles; Tomatine; Tumor Cells, Cultured; Vitamin K

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Topics: Animals; Cell Division; Cell Survival; Colorimetry; Coloring Agents; Concanavalin A; Dose-Response Relationship, Drug; In Vitro Techniques; Interleukin-2; Interleukin-4; Leukemia, Myeloid; Mast Cells; Methylphenazonium Methosulfate; Mice; Spleen; T-Lymphocytes; Tetrazolium Salts; Thiazoles; Vitamin K