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 μM ONOO− and incubated for 4 h, and (2) treated with 10–100 μM ONOO− and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 μM ONOO− and 500 μM AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 μM AsC then exposed to 300 μM 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 μM. Concentrations of ONOO− greater than 300 μM caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO− induced apoptosis at lower concentrations (50;–75 μM), with concentrations > 75 μM 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 © 1996 Elsevier Science Inc.