Detailed analysis of reactive oxygen species induced by visible light in various cell types

Background and Objective: Light in the visible and near infrared region stimulates various cellular processes, and thus has been used for therapeutic purposes. One of the proposed mechanisms is based on cellular production of reactive oxygen species (ROS) in response to illumination. In the present study, we followed visible light (VL)-induced hydroxyl radicals in various cell types and cellular sites using the electron paramagnetic resonance (EPR) spin-trapping technique. Materials and Methods: Fibroblasts, sperm cells, cardiomyocytes, and skeletal muscle cells were irradiated with broadband (400-800 nm) VL. To detect ROS, the EPR spin-trapping technique coupled with the spin-traps 5,5-dimethyl pyrroline-N-oxide (DMPO) or 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) were used. To investigate the cellular sites of ROS formation, the cell-permeable molecule, isopropanol, or the nonpermeable proteins, bovine serum albumin (BSA) and superoxide dismutase (SOD), were introduced to the cells before irradiation. ROS production in mitochondria was measured using the fluorescent probe, MitoTracker Red (MTR). Results and Conclusions: The concentration of ^•OH increased both with illumination time and with cell concentration, and decreased when N ₂ was bubbled into the cell culture, suggesting that VL initiates a photochemical reaction via endogenous photosensitizers. VL was found to stimulate ROS generation both in membrane and cytoplasm. In addition, fluorescent measurments confirmed the mitochondria to be target for light-cell interaction. The findings support the hypothesis that ROS are generated in various cellular sites following light illumination.