Enhanced generation of mitochondrial reactive oxygen species in cybrids containing 4977-bp mitochondrial DNA deletion

The poor bioenergetic state in mitochondria containing mtDNA with the 4977-bp deletion has been well documented. However, information on mitochondrial reactive oxygen species (ROS) generation at rest or under intense oxidative stress in mitochondria lacking the 4977-bp mtDNA fragment inside intact living cells was insufficient. We used cybrids containing truncated mtDNA lacking the 4977-bp fragment and measured ROS levels inside cybrids by fluorescence probe, 2′,7′-dichlorodihydrofluorescein (DCF), and confocal microscopy. Mitochondrial ROS at resting state was slightly higher in cybrids containing 4977-bp deletion mtDNA as compared to cybrids without mtDNA defects. For intense oxidative stress treatment, cybrids were treated with 5 mM H₂O ₂ for 10 min. Consecutive DCF images were acquired after H ₂O₂ had been washed away. Progressive increase of DCF signals, especially in the mitochondrial area, was observed in cybrids containing 4977-bp deletion mtDNA, even long after the brief, intense H ₂O₂ treatment. This result suggests that a feed-forward, self-accelerating vicious cycle of mitochondrial ROS production could be initiated in cybrids containing 4977-bp deletion fragment mitochondria after brief, intense H₂O₂ treatment. This mechanism may play an important role in the pathophysiology of the disease process caused by mitochondria containing mtDNA with the 4977-bp deletion.