Abstract
765
Oxidative DNA damage induced by activated neutrophils is suggested to play a role in the observed association between chronic inflammation and carcinogenesis. H2O2 has been shown to be a crucial factor in neutrophil-induced DNA strand breakage. In normal conditions however, H2O2 will be largely consumed by the neutrophilic enzyme myeloperoxidase (MPO) to form the highly microbicidal compound HOCl. Recently it was shown that nitrite, which is present at sites of inflammation, represents an excellent MPO inhibitor. Since MPO inhibition increases the availability of H2O2, we hypothesized that the presence of nitrite can increase H2O2-mediated DNA strand breakage by neutrophils. Therefore, phorbol ester-activated neutrophils were co-incubated with rat alveolar epithelial cells (RLE) in the presence of nitrite (0-100 μM) or the specific MPO inhibitor 4-amino benzoic acid hydrazide (4-ABAH, 0-100 μM). DNA strand breakage was specifically evaluated in the RLE cells using single cell gel electrophoresis. H2O2 release and MPO activity were determined spectrophotometrically. Both nitrite and 4-ABAH were found to inhibit MPO activity in a dose-dependent manner (40% at 100 μM nitrite, 100% at 100 μM 4-ABAH), and this lead to a significant increase in oxidative DNA damage (140% at 100 μM nitrite; 300% at 100 μM 4-ABAH). The effects were observed in the absence of cytotoxicity (trypan blue) or pH changes. Both nitrite and 4-ABAH did not have an effect on H2O2 production by neutrophils or on DNA damage induced by reagent H2O2 (25 μM). Moreover, incubation with 4-ABAH and nitrite alone did not cause DNA damage in RLE cells. To further evaluate the underlying mechanisms, in vitro experiments were performed in which H2O2 (25 μM) was allowed to react with purified MPO before addition to the RLE cells. In the presence of MPO all H2O2 was consumed, and no subsequent cellular DNA damage could be detected. However, when nitrite (100 μM) was added to these pre-incubations, DNA damage in the RLE cells was comparable with levels as induced by H2O2 alone. Inhibitory experiments with catalase further confirmed that these effects are H2O2-dependent, indicating nitrite-induced inhibition of MPO activity. In conclusion, the present data demonstrate that physiological levels of nitrite can enhance the DNA damaging capacity of neutrophils. This effect is likely due to nitrite-induced inhibition of MPO, which increases the availability of H2O2. Further studies are needed to assess the implication of our current observations for in vivo DNA damage in relation to neutrophilic inflammation.
- American Association for Cancer Research