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

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/UHDR irradiation 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 versus conventional radiotherapy (CONV-RT) in murine glioma GL261 cells and primary healthy astrocytic cultures, focusing on redox dynamics, mitochondrial function, bioenergetics, and stress transcriptional 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 promoted relative recovery of mitochondrial membrane potential and respiratory capacity compared to CONV-RT. FLASH irradiation was also associated with sustained induction of cell-cycle checkpoint and senescence-related markers. 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 apoptosis between FLASH- and CONV-RT, consistent with tumor responses being associated with checkpoint-dependent proliferative constraint and metabolic dysfunction rather than enhanced apoptotic execution. Pharmacological inhibition of PARP, calpains, necroptosis, and caspases differentially modulated mitochondrial bioenergetic parameters across modalities and cell types, supporting pathway-diverse contributions to radiation-induced metabolic stress. Tumor-selective stress responses were also observed in human U87 glioblastoma cells, where FLASH irradiation induced stronger ROS accumulation, enhanced apoptotic engagement, 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 reprogramming that was associated with relative preservation of mitochondrial function in healthy astrocytes while imposing prolonged metabolic and proliferative constraints in glioma cells, highlighting distinct early redox-mitochondrial adaptations induced by FLASH/UHDR irradiation in astrocytic and glioma models.