Genotoxicity analysis
DNA DAMAGE, LYSIS
DNA damage was detected through alkaline single cell gel electrophoresis (Comet assay). Cell suspensions were mixed with 0.5% low-melting point agarose (VWR) and seeded onto glass slides coated with 1% normal agarose (VWR) in duplicate. Slides were incubated with lysis buffer (2.5 M NaCl, 100 mM NaCell nuclei were stained with ethidium bromide (20 µg/mL) and imaged using an Olympus BX51 microscope and Olympus DP71 camera under a 20 × objective. Comets were analysed using the free comet analysis software Casplab_1.2.3b2 (CaspLab.com). For each duplicate, 50 and 100 cells were analysed for in vitro, and lavage cells (BW and BAL), respectively. OTM [(tail mean–head mean) x tail% DNA/100)] was calculated and used in statistical analyses. All samples from each individual subject were analysed simultaneously to minimise inter-assay variation.