linoleic-acid and thiazolyl-blue

Antioxidants scavenge free radicals, singlet oxygen, and electrons in cellular redox reactions. The yellow MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] is reduced to a purple formazan by mitochondrial enzymes. NADPH is the basis of established in vitro cell viability assays. An antioxidant assay has been developed utilizing the redox reaction between MTT and selected natural product extracts and purified compounds. This simple, fast, and inexpensive MTT antioxidant assay is comparable with the lipid peroxidation inhibitory assay and can be mechanized to achieve high throughput.

Topics: Antioxidants; Coloring Agents; Formazans; Free Radical Scavengers; Mitochondria; Molecular Structure; NADP; Oxidation-Reduction; Plant Extracts; Singlet Oxygen; Tetrazolium Salts; Thiazoles

In this study, we investigated the extent of the cytotoxicity effect of oxidised lipids and whether tea catechins namely (-)epigallocatechin-3 gallate (EGCG) decreased lipid peroxidation in caco-2 cells. Cells treated with 0-100 microg/ml fish oil or methyl linoleate (ML) oxidised by UV irradiation for 24 h, 48 h and 72 h, indicated a substantial decrease in cell viability especially in samples treated with 100 microg/ml oxidised lipid. Addition of malondialdehyde (MDA) and hydroxynonenal (50 microM) also reduced cell viability. Using EGCG (50 microM) increased the viability of cells treated with 24 h oxidised mackerel oil (72% live and 28% dead) compared with 48 h oxidised mackerel oil (89% live and 11% dead) and 72 h oxidised mackerel oil (71% live and 29% dead) as monitored by the MTT assay. Apoptosis of caco-2 cells by oxidised fish oil and ML and protection by EGCG was confirmed using fluorescence microscopy and caspase-3 presence by Western blotting.

Topics: Antineoplastic Agents; Apoptosis; Bisbenzimidazole; Blotting, Western; Caco-2 Cells; Caspase 3; Catechin; Coloring Agents; DNA, Neoplasm; Fish Oils; Fluorescent Antibody Technique; Humans; Linoleic Acid; Lipid Peroxidation; Lipids; Oxidation-Reduction; Tea; Tetrazolium Salts; Thiazoles; Thiobarbituric Acid Reactive Substances; Ultraviolet Rays

Conversion of 12,13-cis-epoxyoctadecenoic acid (12,13-EOA) to 12,13-dihydroxyoctadecenoic acid (12,13-DHOA) by soluble epoxide hydrolase has been suggested to be a critical step in mediating the toxicity of epoxidized linoleic acid. The current study tests the hypothesis that low levels of albumin in the normal culturing media of Sf-21 cells can protect these cells from exposures to 12,13-EOA, but not 12,13-DHOA. In albumin-free media, Sf-21 cells exposed to 100 microM 12,13-EOA, and 12,13-DHOA for 1 min showed significant signs of mitochondrial dysfunction which led to cytotoxicity. The addition of bovine serum albumin (BSA) at a concentration (3 microM) found in normal serum-supplemented media protected Sf-21 cells exposed to 12,13-EOA, but not 12,13-DHOA while BSA (500 microM) fully protected Sf-21 cells exposed to these fatty acids. These data resolve previous discrepancies observed among in vitro models and help clarify our understanding of how these metabolites affect human health.

Topics: Animals; Cattle; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Humans; Linoleic Acid; Mitochondria; Oleic Acids; Oxygen; Oxygen Consumption; Serum Albumin, Bovine; Spodoptera; Tetrazolium Salts; Thiazoles

Amitriptyline and imipramine, two tricyclic antidepressant drugs, have been studied to evaluate their phototoxic potential using various models. Reactive oxygen species production was investigated. A negligible production of singlet oxygen was observed for both compounds whereas a significant production of superoxide anion was noted for amitriptyline in particular. Moderate red blood cell lysis under UVA light (365 nm) was induced in the presence of the two drugs at a concentration of 50 microM. Cellular phototoxicity was investigated on a murine fibroblast cell line (3T3). The two drugs were phototoxic causing cell death at a concentration of 100 microM and a UVA dose in the range of 3.3-6.6 J/cm2. Furthermore, the two drugs photosensitized the peroxidation of linoleic acid, as monitored by the formation of dienic hydroperoxides. The presence of BHA and GSH, two free radical scavengers, significantly reduced the lipid oxidation photoinduced by the drugs, suggesting a predominant involvement of radical species. Finally, the involvement of nucleic acids in the phototoxicity mechanism was also investigated using a pBR322 plasmid DNA as a model.

Topics: Amitriptyline; Animals; Antidepressive Agents, Tricyclic; Cells, Cultured; Chromatography, High Pressure Liquid; Coloring Agents; Dermatitis, Phototoxic; DNA; DNA Damage; Erythrocytes; Fibroblasts; Hemolysis; Imipramine; Linoleic Acid; Lipid Peroxidation; Mice; Mice, Inbred BALB C; Photochemistry; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Ultraviolet Rays