Fig. 1. Time-dependent formation of ROS and oxidative DNA damage in HCEC and CRC cells by heme iron versus inorganic iron.

a, c HCEC were incubated for 30 min (a) or 2 h (c) with increasing doses of hemin or FeCl₃ (0–200 µM). Reactive oxygen species (ROS) levels were assessed by live cell staining and subsequent flow cytometry-based analysis. b, d HCT116 were treated for 30 min (b) or 2 h (d) and analyzed as described under a. Data (a–d) is shown as mean + SEM (n ≥ 3, except for 20 µM in HCT116 at 2 h: n = 2). Ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 (versus respective control). e, f Cells were exposed to hemin (0–200 µM) or FeCl₃ (200 µM) for 2 h. DNA strand break induction and formation of oxidative DNA damage was determined by the alkaline Comet assay without (e) or with Fpg (f). Data are presented as mean + SEM (n ≥ 3). Ns: p > 0.05; *p < 0.05; **p < 0.01 (versus control).