ROS production and oxidative DNA damage in smokeless tobacco extracts – exposed fibroblasts. Intracellular ROS levels were measured by carboxy-H2DCFDA fluorescence and flow cytometry after exposure to 0 (Ctr), 0.1%, 1%, 2%, or 4% of Tob for 2 h, 24 h, or 6 d, or 2 h followed by 24 h recovery in Tob-free medium (A). Oxidative DNA damage was measured using the Fpg-FLARE comet assay and depicted as the average comet tail (B) and the distribution of normalized mean tail lengths (C). Tail lengths were normalized by subtracting the mean tail length of the untreated sample from that of the Fpg-treated sample. At least 50 randomly chosen comet tails were analyzed per duplicate sample.