Chronic inhalation of environmental particles is associated with pulmonary carcinogenesis. Although the mechanism has not yet been fully elucidated, influx of inflammatory cells, including neutrophils, is suggested to play a major role in this process. Typically, in the particle-exposed lung, influx of neutrophils is accompanied by an accumulation of nitrite. Previous studies indicated that nitrite may affect the toxicity of neutrophils, involving an interaction with neutrophil-derived myeloperoxidase (MPO). To evaluate the possible consequences of this interaction for inflammation-mediated genotoxicity, we investigated the effect of nitrite on neutrophil-induced DNA damage in pulmonary target cells. Therefore, activated neutrophils were co-cultured with alveolar type II epithelial cells (RLE), and DNA strand breakage was evaluated using single-cell gel electrophoresis (comet assay). In this system, addition of nitrite caused an increase in neutrophil-induced DNA strand breakage in RLE cells, which was associated with an inhibition of MPO activity. Similar results were obtained by co-culturing RLE cells with neutrophils in the presence of the specific MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). To further investigate the mechanism underlying these observations, in vitro experiments were performed using mixtures of nitrite, MPO and its substrate H2O2. DNA strand breakage by reagent H2O2 was inhibited when it was allowed to react with MPO before addition to the RLE cells. However, when MPO and H2O2 were pre-mixed in the presence of nitrite or 4-ABAH, the inhibitory effect of MPO on resultant DNA damage was reversed. Further studies using catalase indicated that DNA strand breakage by the pre-mixtures of MPO, H2O2 and nitrite was H2O2-specific, suggesting that nitrite prevents consumption of H2O2 by MPO. Collectively, our results show that nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells. This effect is probably due to an inhibition of MPO activity, which increases the availability of its DNA strand breaking substrate H2O2.
Knaapen, A. M., Schins, R. P., Borm, P. J. A., & van Schooten, F. J. (2005). Nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells by inhibition of myeloperoxidase. Carcinogenesis, 26, 1642-1648. https://doi.org/10.1093/carcin/bgi116