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

Abstract

Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous 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 measured changes of mitochondrial calcium level ([Ca(2+)](m)) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca(2+)](m) level changes. Changes of intracellular ROS and [Ca(2+)](m) were visualized using fluorescent probes 2',7'-dichlorodihydrofluorescein (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 [Ca(2+)](m) was also induced by photoirradiation. 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 Ca(2+) levels were also high. In cells that had mild ROS levels, mitochondrial Ca(2+) 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 Ca(2+) levels in astrocytes irradiated by visible light.

Dual Phases of Respiration Chain Defect-Augmented mROS-Mediated mCa2+Stress during Oxidative Insult in Normal andρ0RBA1 Astrocytes

“…In the current study, we found that mROS generation is prior to mCa 2+ increase in control and ρ 0 astrocytes after H 2 O 2 treatment. These findings were compatible with our previous studies on H 2 O 2 [32] and photoirradiation-augmented oxidative stress [33–35]. Moreover, our study provided quantitative imaging evidence that dual effect on antioxidation exists in ρ 0 astrocytes during different extent of oxidative stress.…”

Cargo-Free Nanoparticles Containing Cationic Lipids Induce Reactive Oxygen Species and Cell Death in HepG2 Cells

“…Several mechanisms explaining ROS generation have been suggested in mitochondria, which are associated with mitochondrial membrane potential, nitric oxide, and Ca 2+ influx. 31,32) Recently, Sun et al suggested that NADPH oxidase activation is an initiator of mitochondrial damage, which eventually results in the death of HepG2 cells. 33) Based on these results, we also measured the enzyme activity after treatment with cationic NPs used in this study.…”

Zinc is a novel intracellular second messenger

“…Fluorescence laser scanning is known to induce reactive oxygen species (Jou et al, 2004), which may induce the release of free zinc from zinc-binding proteins such as metallothionein, independent of receptor-mediated signaling events. To identify the subcellular regions where the zinc wave originates while avoiding secondary effects caused by cell stress, we used a thin-layer illumination microscope system that was based on the total internal reflection fluorescence (TIRF) microscope (Tokunaga et al, 1997).…”