The Use of the single cell gel electrophoresis assay in detecting DNAsingle strand breaks in lung cells in vitro

Abstract

DNA single strand breaks (SSB) can be used as a biomarker ofoxidant exposure, and also as an indicator of the carcinogenicity/mutagenicity of a substance. The single cell gel electrophoresis (SCGE) assay is more sensitive and requires fewer cells compared to other techniques used for detecting SSB. We examined the utility of using the SCGE assay for human lung cells exposed to endogenous and exogenous oxidants. A human bronchial cell line (BEAS) was used as a model of airway epithelial cells in this study. BEAS cells exposed to 0–50 μM hydrogen peroxide (H2O2) for 60 min at 4°C exhibited a concentration-dependent increase in SSB as determined by an increased DNA migration area in a gel undergoing electrophoresis. H2O2-induced increases in DNA SSB were also demonstrated using cultured normal human tracheobronchial epithelial (NHBE) cells and human alveolar macrophages in a concentration response manner. BEAS cells were also exposed to air or ozone (O3) on a Transwell filter without medium present apically. Cells exposed to O3 at 0.1 or 0.4 ppm at 37°C for up 120 min had a time- and concentration-dependent increase in SSB compared to air-exposed cells. NHBE cells exposed to 0.4 ppm 03 (60 min) also had increased DNA SSB. Cells with H2O2-induced DNA SSB can be frozen and stored up to 4 weeks without altering the original DNA SSB. These findings indicate that SC GE can be used to detect SSB in cultured lung cells, and has applicability for detecting SSB in lung cells recovered from in vivo and in vitro exposures to oxidants.