BACKGROUND: Peroxynitrite (ONOO-) is a potent oxidant that is readily formed from the interaction of nitric oxide and superoxide. Recent evidence suggests that the harmful effects of nitric oxide on biological sytems may result from its conversion to other reactive nitrogen intermediates, with ONOO-being a potential candidate. Our goal was to determine if peroxynitrite initiated cell death by apoptosis in human epithelial cells and to evaluate the effects of antioxidants. METHODS: Cultured human epithelial cells (T84), grown to confluence, were treated with ONOO- (25-300μM) in the presence or absence of ascorbic acid (500μM) or 5-aminosalicylic acid (5-ASA, 10-200μM). Necrosis was determined by the failure to exclude trypan blue and apoptosis was evaluated by flow cytommetry, TUNEL immunohistochemistry and ELISA for cytosolic internucleosomal DNA fragments. Peroxynitrite was formed on site from sodium nitrite, hydrogen peroxide and nitric acid. Unreacted peroxide was eliminated by manganese oxide and decomposed peroxynitrite was used as a control. RESULTS: Low concentrations of ONOO- (25-300μM) elicited a delayed cell death that bore the hallmarks of apoptosis. The degree of apoptosis increased with time after exposure (4-14h). High doses of ONOO- elicited a more immediate, necrotic cell death. Ascorbic acid significantly(p<0.05) reduced T84 apoptosis either with co-incubation or pretreatment followed by washout prior to ONOO- exposure. 5-ASA was considerably more potent than ascorbic acid; the IC50 for the prevention of apoptosis was 16μM. Both 5-ASA and ascorbic acid facilitated the chemical decomposition of ONOO-, determined spectrophotometrically. However, neither antioxidant modified NO degradation in buffer solutions. CONCLUSION: Exposure of epithelial cells to low doses of peroxynitrite causes apoptosis. Ascorbic acid and 5-ASA prevent this response via intracellular and extracellular mechanisms without interacting with the precursor NO. Supplemental antioxidants should be considered in NOx mediated pathology.