Quantitation of intracellular oxidation in a renal epithelial cell line

We quantitated the presence of intracellular oxidizing species in response to oxidative stimuli using fluorescent cell analytic techniques. The studies were performed with a laser-activated flow cytometry system using 2',7'-dichlorofluorescin diacetate (DCFDA) as a probe for intracellular oxidation events. Oxygen radical formation was initiated by the addition of FeCl2 or xanthine oxidase to the culture media. Xanthine oxidase and FeCl2 both increased intracellular DCFDA oxidation over control (p less than .001). Increases in intracellular DCFDA oxidation in response to xanthine oxidase exposure were inhibited by extracellular superoxide dismutase, catalase and dimethyl sulfoxide (p less than 0.001), implicating the superoxide anion, hydrogen peroxide, and the hydroxyl radical in producing the changes in intracellular dichlorofluorescein fluorescence. Increases in intracellular DCFDA oxidation in response to xanthine oxidase correlated with loss of cellular viability, as established by decreased plating efficiency. We conclude that relative intracellular oxidation can be quantitated within the cultured renal cell and that some extracellularly generated radicals may be capable of traversing the intact cell membrane to oxidize DCFDA in the cell interior.