Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells: Attenuation by L-ascorbic acid

The free radicals nitric oxide and superoxide react to form peroxynitrite (ONOO-), a potent cytotoxic oxidant. This study was designed to evaluate whether addition of L-Ascorbic acid (AsC) into the culture medium decreases peroxynitrite-induced apoptosis in human intestinal epithelial (T84) and murine macrophage (RAW 264.7) cell lines. In Experiment 1, T84 and RAW 264.7 cells were divided in two protocols: (1) treated with 100-300 mu M ONOO- and incubated for 4 h, and (2) treated with 10-100 mu M ONOO- and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 mu M ONOO- and 500 mu M AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 mu M AsC then exposed to 300 mu M ONOO- for 4 h. Cell viability (necrosis) was assessed by trypan blue dye exclusion. Apoptosis was quantified with a cell death detection ELISA assay. In the 4 h protocol, ONOO- induced apoptosis in T84 and RAW 264.7 cells, at levels of 100-300 mu M. Concentrations of ONOO- greater than 300 mu M caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO- induced apoptosis at lower concentrations (50; -75 mu M), with concentrations > 75 mu M resulting in necrosis. AsC administered to the media or with preincubation plus washout, decreased peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells. These results indicate that ONOO- may contribute to the pathophysiology of gut inflammation by promoting cell death and ascorbic acid may protect against peroxynitrite-induced damage. Copyright (C) 1996 Elsevier Science Inc.