Differential redox-mitochondrial adaptation to ultra-high-dose-rate irradiation in astrocytes and glioma cells

Abstract FLASH radiotherapy (FLASH-RT) delivers radiation at ultra-high dose rates (UHDRs) and has been reported to reduce normal tissue toxicity while maintaining tumor control, yet the cellular mechanisms underlying the ‘FLASH effect’ remain incompletely defined. FLASH-RT has been associated with differential normal and tumor tissue responses in preclinical studies, although the early cellular processes induced under these conditions remain incompletely understood. Here, we compared FLASH-RT versus conventional radiotherapy (CONV-RT) in murine glioma GL261 cells and primary healthy astrocytic cultures, focusing on redox dynamics, mitochondrial function, bioenergetics, and transcriptional stress programs. Using real-time kinetic assays, flow cytometry, Seahorse analysis, and gene expression profiling, we found that in healthy astrocytes FLASH-RT reduced acute ROS accumulation, limited cell death, and was associated with greater preservation of mitochondrial membrane potential and respiratory capacity compared with CONV-RT. FLASH-RT was also associated with sustained induction of cell-cycle checkpoint and senescence-associated transcripts. In GL261 cells, FLASH-RT produced a lower initial ROS peak followed by continued ROS accumulation over time and enhanced G2/M accumulation, together with persistent mitochondrial depolarization and respiratory suppression. Annexin V analysis at 8 Gy revealed comparable levels of Annexin V positivity between FLASH- and CONV-RT, whereas FLASH-treated cells showed greater G2/M accumulation and metabolic dysfunction, consistent with altered cell-cycle progression rather than enhanced Annexin V-defined cell death. Pharmacological modulation with DPQ, Calpeptin, Nec-1 and zVAD differentially affected mitochondrial bioenergetic parameters across irradiation modalities and cell types, providing functional evidence for the potential contribution of distinct stress- and cell-death-associated signaling pathways to radiation-induced metabolic dysfunction. Similar stress responses were also observed in human U87 glioblastoma cells, where FLASH-RT induced stronger ROS accumulation, greater Annexin V positivity, and alterations in NAD⁺ levels associated with mitochondrial respiratory impairment compared with CONV-RT. Collectively, these data indicate that FLASH-RT elicits cell-type-specific redox–mitochondrial responses associated with relative preservation of mitochondrial function in healthy astrocytes and prolonged metabolic stress and altered cell-cycle dynamics in glioma cells, highlighting distinct early adaptations to FLASH irradiation in astrocytic and glioma models.