Reactive oxygen species in monocytes generated by blue-light activated chromophores

Objectives: Photodynamic therapy using red-light chromophores has been proposed to treat endodontic infections by generating reactive oxygen species (ROS). However, red light significantly heats tissues and increases the risk of thermal trauma to resident cells. In the current study, we tested the hypothesis that several blue light chromophores (380-500 nm) generate intracellular ROS at concentrations that have minimal cytotoxicity to mammalian cells. Methods: Blue light chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) were added for 24 h to cultured THP1 human monocytes (concentrations between 0.1 nM to 100 然). Cytotoxicity was determined with or without blue light activation (QTH, 27 J/cm,²) by measuring mitochondrial activity with the MTT method (n = 3/condition). For ROS measurements, THP1 cells were exposed to 10 然 of chromophores for 30 min before activation with blue light (27 J/cm²). Intracellular ROS were measured for 2 h using a dihydrofluorescein diacetate fluorescence assay. Controls received light or chromophores alone. Positive controls received diamide (5 mM, 30 min). Results: Pheophorbide-a was the most cytotoxic, suppressing cellular SDH activity by 50% (TC50) at concentrations of about 5 然. Pheophorbide-a-PLL was less cytotoxic (TC50 of 10 然). Chlorin e6 had a TC50 of about 30 然, and riboflavin alone showed no cytotoxicity at concentrations <100 然. All chromophores, except pheophorbide-a-PLL were significantly (p < 0.05) more cytotoxic after blue light irradiation. All chromophores produced intracellular ROS levels higher than controls (p < 0.05) that were dependent on both the presence of the photosensitizer and blue light exposure. Riboflavin and pheophorbide-a-PLL produced the highest levels of ROS. Conclusion: Blue light chromophores have possible utility as producers of intracellular ROS that may be useful for endodontic disinfection with minimal eukaryotic cell cytotoxicity.