Mitochondrial Reactive Oxygen Species Generation and Calcium Increase Induced by Visible Light in Astrocytes

Abstract

Mitochondria contain photosensitive chromophores that can be ac-tivated or inhibited by light in the visible range. Rather than utilizing light en-ergy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light Our pre-vious work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation-induced phototoxicity and apoptosis. In this study, we mea-sured changes of mitochondrial calcium level ([Ca²⁺]_m) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca²⁺]_m level changes. Changes of mtracellular ROS and [Ca²⁺]_m were visualized using fluorescent probes 2′,7′-dichlorodihy-drofluorescein (DCF), and rhod-2 After exposure to visible light irradiation, RBA-1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca²⁺]_m was also induced by photo-irradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca²⁺ levels were also high. In cells that had mild ROS levels, mitochondrial Ca²⁺ levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod-2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca²⁺ levels in astrocytes irradiated by visible light.