Antioxidants React with Higher Oxides of Nitrogen but Not Nitric Oxide: Therapeutic Implications

Abstract 1840 Poster Session I, Saturday, 5/1 (poster 87)

INTRODUCTION: While nitric oxide (NO) has anti-inflammatory properties under basal conditions, where production is low. However, it remains enigmatic as to why NO displays pro-inflammatory characteristics in chronic inflammation; conditions where NO production is elevated. From this we must either assume that the anti-inflammatory actions are weak and of little consequence, or alternatively other factors influence the role of NO in chronic inflammation. We hypothesized that the answer to this enigma may lie in the conversion of NO to other, higher oxides of nitrogen (NO₂, N₂O₃, ONOO^-) which are far more reactive than NO itself. To address this we used antioxidants to negate the levels of the nitrogen oxides and their cellular consequences as well as testing for inherent reactivity between the species. METHODS: NO levels were monitored electrochemically using a microelectrode that selectively measured NO levels in solution. Antioxidants were incubated with NO in the presence and absence of oxygen. The rate of NO degradation (in the presence of oxygen) was determined along with spectral analysis of the antioxidant. Reactivity with peroxynitrite was assessed with freshly prepared stock at either pH 13 or pH 7.4. Antioxidants were added to cell culture media to assess their effects on peroxynitrite-induced apoptosis. The following antioxidants were tested: histidine, homocysteine, ascorbic acid and 5-aminosalicylic acid. RESULTS: Antioxidant UV absorbance was modified by NO only in the presence of oxygen, indicating that NO must be oxidized in order for it to be reactive. None of the antioxidants promoted the degradation of NO, as determined electrochemically. Even in the presence of oxygen, the decay curves of NO were identical in the presence and absence of antioxidant (remaining second order), indicating that the initial reaction of NO and O₂ was unaffected. The one exception was ascorbic acid, which attenuated the oxidative degradation of NO but retained its second order characteristics. This indicates that ascorbic acid recycled NO downstream from the initial reaction of NO and O₂. All the antioxidants tested degraded peroxynitrite and attenuated peroxynitrite-induced apoptosis of human epithelial cells and mouse macrophages. CONCLUSION: Despite being a free radical NO does not react with antioxidants. Antioxidants do react with oxidative products of NO (NO₂ and N₂O₃) and peroxynitrite and reduce their toxicity. Thus, antioxidants can be utilized to negate the pro-inflammatory consequences of elevated NO synthesis independent of interactions with NO itself.