ABSTRACT
The sensitive single-cell analytical techniques of flow cytometry and propidium iodide-binding have been used to examine the molecular effect of oxidative stress on cultured human skin fibroblasts. Cells synchronized by limited time attachment were exposed to a hypoxanthine-xanthine oxidase (HX/XO) system at different intervals after subculture. The characteristic feature of the treated population was a variation of the amount of nuclear DNA propidium iodide (PI)-fluorescence staining. Increased fluorescence intensity was observed with a shift of the G1/G0 and G2/M peak, which is dependent on both cell cycle stage and treatment level. When scavenger molecules (catalase, silybin) were added to the oxidative reaction, the nuclear DNA histogram of HX/XO-treated cells was similar to that obtained from untreated cells. In parallel, UV absorbance studies in vitro have shown that PI is capable of binding extensively to DNA when isolated from HX/XO-exposed cells, compared with control cells or HX/XO-exposed cells in the presence of scavengers. These results indicate that free radicals are responsible for the increase in fluorescence intensity in the HX/XO-exposed cells. This change in DNA stainability would be due to an opening of the DNA strands in situ, leading to an unmasking of new PI-binding sites on DNA. The strand separation may facilitate access on the fluorochrome to DNA, thereby enhancing dye binding. This flow cytometric assay based on DNA biophysical changes caused by free radicals is a useful means of measuring pro- and/or anti-oxidant potential.
