oxypurinol and thiazolyl-blue

In HeLa cells evidence is provided that active oxygen species such as hydrogen peroxide and superoxide at low levels are important growth regulatory signals. They may constitute a novel regulatory redox system of control superimposed upon the established cell growth signal transduction pathways. Whilst for example hydrogen peroxide can be added exogenously to elicit growth responses in these cells, it is clear that cellularly generated superoxide and hydrogen peroxide are important. Experiments with superoxide dismutase, superoxide dismutase mimics and inhibitors of both superoxide dismutase and xanthine oxidase suggest that superoxide generated intracellularly and superoxide released extracellularly are both relevant to growth control in HeLa cells.

Topics: Allopurinol; Catalase; Cell Division; Ditiocarb; HeLa Cells; Humans; Hydrogen Peroxide; Oxidation-Reduction; Oxypurinol; Reactive Oxygen Species; Salicylates; Signal Transduction; Superoxide Dismutase; Superoxides; Tetrazolium Salts; Thiazoles; Xanthine Oxidase

Experiments have been carried out to explore the use of a tetrazolium salt, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in the detection of intracellularly generated superoxide in HeLa cells. From the use of a low molecular weight lipophilic mimic of superoxide dismutase, as well as superoxide dismutase, and inhibitors of superoxide dismutase, it is suggested that at least 20-30% of the intracellular reduction of MTT is due to superoxide. Whilst this may arise from mitochondria another possible intracellular source in HeLa cells may be xanthine oxidase. The overall rate of intracellular MTT reduction in HeLa cells is inversely dependent on levels of serum in the culture medium. Serum components with a modulatory role in this context are those with antioxidant function. Reduced MTT is also detectable extracellularly in cultures of HeLa cells and at least 80% of this is due to superoxide. Use of inhibitors suggest that whilst a small proportion (30%) may arise through an NADPH-oxidase type enzyme, other sources of extracellular superoxide in HeLa cells remain a possibility.

Topics: Allopurinol; Anti-Infective Agents; Biphenyl Compounds; Coloring Agents; Culture Media; Culture Media, Serum-Free; Formazans; HeLa Cells; Humans; Kinetics; Onium Compounds; Oxidation-Reduction; Oxypurinol; Superoxide Dismutase; Superoxides; Tetrazolium Salts; Thiazoles; Vitamin E