OXIDATIVE STRESS-INDUCED APOPTOSIS PREVENTED BY TROLOX

The ability of oxidative stress to induce apoptosis (programmed cell d eath), and the effect of Trolox, a water soluble vitamin E analog, on this induction were studied in vitro in mouse thymocytes. Cells were e xposed to oxidative stress by treating them with 0.5-10 mu M hydrogen peroxide (H2O2) for 10 min, in phosphate-buffered saline supplemented with 0.1 mM ferrous sulfate. Cells were resuspended in RPMI 1640 mediu m with 10% serum and incubated at 37 degrees C under 5% CO2 in air. El ectron microscopic studies revealed morphological changes characterist ic of apoptosis in H2O2-treated cells. H2O2 treatment fragmented the D NA in a manner typical of apoptotic cells, producing a ladder pattern of 200 base pair increments upon agarose gel electrophoresis. The perc entage of DNA fragmentation (determined fluorometrically) increased wi th increasing doses of H2O2 and postexposure incubation times. Pre- or posttreatment of cells with Trolox reduced H2O2-induced DNA fragmenta tion to control levels and below. The results indicate that oxidative stress induces apoptosis in thymocytes, and this induction can be prev ented by Trolox, a powerful inhibitor of membrane damage.