Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells

Abstract

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 ONOOmay contribute to the pathophysiology of gut inflammation by promoting cell death and ascorbic acid may protect against peroxynitrite-induced damage.