Mitochondrial hOGG1-2a overexpression sensitizes HeLaS3 cells to oxidative stress, accompanied by mitochondrial dysfunction and reduced mtDNA integrity.

Mitochondria are essential organelles responsible for cellular ATP production and contain their own mitochondrial DNA (mtDNA), which encodes key components of oxidative phosphorylation. Because mitochondria continuously generate reactive oxygen species (ROS), mtDNA is particularly susceptible to oxidative damage. Although DNA repair enzymes are present in mitochondria, the regulation of mtDNA repair and its impact on cellular responses to oxidative stress remain incompletely understood. Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key enzyme in the base excision repair (BER) pathway, and the mitochondrial isoform hOGG1-2a contributes to the maintenance of mtDNA integrity.In this study, HeLaS3 cell lines stably overexpressing hOGG1-2a were established to examine responses to oxidative stress. hOGG1-2a overexpression was associated with reduced survival following H2O2 treatment, γ-ray exposure, heat shock, and ultraviolet C (UVC) irradiation. Apoptotic cell death increased after oxidative stress. Mitochondrial membrane potential assessed by JC-1 staining was significantly reduced in hOGG1-2a-overexpressing cells. Long-range PCR analysis revealed reduced mtDNA amplification efficiency, and oxidative stress was accompanied by a greater reduction of the mitochondrial enzyme Aconitase 2. These cells exhibited elevated basal ATP levels and altered ATP responses under oxidative stress conditions. In addition, mitochondrial superoxide-associated fluorescence detected by MitoSOX™ was significantly increased.Combined long-range PCR and Sanger sequencing indicated reduced mtDNA amplification after H2O2 exposure without a marked increase in point mutations.Collectively, these findings suggest that hOGG1-2a overexpression sensitizes cells to oxidative stress and is associated with mitochondrial redox dysregulation, reduced mitochondrial membrane potential, altered ATP responses, and reduced mtDNA amplifiability during prolonged stress.