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 microM ONOO- and incubated for 4 h, and (2) treated with 10-100 microM ONOO- and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 microM ONOO- and 500 microM AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 microM AsC then exposed to 300 microM 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 microM. Concentrations of ONOO- greater than 300 microM caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO- induced apoptosis at lower concentrations (50;-75 microM), with concentrations > 75 microM 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 peroxynitrie-induced damage.
Sandoval
M
M
Department of Pediatrics, Louisiana State University School of Medicine, New Orleans 70112, USA.
Zhang
X J
XJ
Liu
X
X
Mannick
E E
EE
Clark
D A
DA
Miller
M J
MJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media; D004848:Epithelium; D016166:Free Radical Scavengers; D006801:Humans; D007422:Intestines; D008264:Macrophages; D051379:Mice; D009336:Necrosis; D009566:Nitrates / Q000494:pharmacology*; D014343:Trypan Blue
D003470:Culture Media; D016166:Free Radical Scavengers; D009566:Nitrates; C039642:peroxynitric acid; D014343:Trypan Blue; D001205:Ascorbic Acid