Figure 4.

ROS formation and mitochondrial DNA damage in hemin-treated cells. (a and b) Cellular ROS levels in differentiated SH-SY5Y cells in the presence or absence of hemin (5 μM) was assessed using the H₂O₂ sensor pHyPer. This sensor emits green fluorescence in the presence of ROS, allowing detection by IF. pHyper expression vectors with targeting sequences for the cytosol, mitochondria, or nuclei were stably transfected in SH-SY5Y cells. Hemin-induced ROS accumulation in mitochondria occurred very early at 5 min and at a significantly higher level than the cytosol or nucleus. Nuclear ROS accumulation had slower kinetics. The histogram represents quantitation of GFP fluorescence intensity from 10 different fields. Scale bar, 20 μm. (c) Loss of mitochondrial membrane potential in differentiated neurons treated with hemin (5 or 10 μM, 1 h) compared to control cells. (d and e) Analysis of DNA integrity measured by LA-PCR of mitochondrial DNA extracted from differentiated neurons treated with hemin or iron (5 or 10 μM) for 12 h. Picogreen-based quantitation of amplified DNA expressed as percent (%) of DNA integrity. *p < 0.01, **p < 0.05. Three independent experiments were performed, and the results are represented as mean ± SEM. Scale bar, 10 μm.