REDUCTION OF A TETRAZOLIUM SALT AND SUPEROXIDE GENERATION IN HUMAN TUMOR-CELLS (HELA)

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.