GSTM3 alleviates FLASH X-ray-induced testicular injury by modulating the ferroptosis pathway

  • Radiother Oncol. 2026 Jun 24:222:111667. doi: 10.1016/j.radonc.2026.111667.
Xiaoyu Zhi  1 ,  Lehui Du  2 ,  Xiang Huang  2 ,  Fang Liu  2 ,  Na Ma  2 ,  Xingdong Guo  2 ,  Yuan Wang  2 ,  Qiduo He  2 ,  Wei Yu  2 ,  Xiangkun Dai  2 ,  Jianxin Wang  3 ,  Xianhong Liu  4 ,  Dai Wu  3 ,  Pei Zhang  5 ,  Xiao Lei  6 ,  Baolin Qu  7
Affiliations
  • 1. The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing, China; Medical School of Chinese PLA, Beijing, China.
  • 2. The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing, China.
  • 3. Institute of Applied Electronics, China Academy of Engineering Physics, Mianyang, Sichuan, China.
  • 4. Zhongjiu Flash Medical Technology Co., Ltd, Mianyang, Sichuan, China.
  • 5. The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing, China. Electronic address: [email protected].
  • 6. The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing, China. Electronic address: [email protected].
  • 7. The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing, China. Electronic address: [email protected].
PMID: 42342042 DOI: 10.1016/j.radonc.2026.111667
Abstract

Background and purpose: Although X-ray FLASH radiotherapy (FLASH-RT) has shown promise in reducing normal tissue toxicity, its effects on the testis and the underlying mechanisms remain poorly understood. This study aimed to investigate the characteristics and mechanisms of X-ray FLASH-RT-induced testicular injury in C57BL/6J mice.

Methods: Testicular injury was evaluated following FLASH-RT at different doses (0, 2, 6, 8, 12, and 20 Gy) and time points (days 1, 7, 21, and 70), with conventional radiotherapy (CONV-RT) as a comparator. Histological and functional damage was assessed by hematoxylin and eosin staining, Ki-67 immunostaining, TUNEL staining, and epididymal sperm counts. Testicular tissues collected on day 7 after irradiation were subjected to RNA Sequencing and proteomic analysis. The role of GSTM3 in response to FLASH-RT and CONV-RT was validated in mouse testes and in the GC-1 mouse spermatogonia cell line. Ferroptosis was evaluated by detecting ferroptosis-related proteins and ultrastructural changes using transmission electron microscopy. In addition, a FLASH-RT-resistant GC-1 cell line (GC-1R) was established and analyzed by single-cell RNA Sequencing (scRNA-seq).

Results: FLASH-RT-induced testicular injury exhibited dose- and time-dependent features. On day 7 after 6 Gy irradiation, FLASH-RT caused less histological damage than CONV-RT. Integrated transcriptomic and proteomic analyses implicated Ferroptosis in this process and identified GSTM3 as a FLASH-RT-responsive molecule. Functional experiments showed that GSTM3 downregulation aggravated FLASH-RT-induced testicular injury, whereas GSTM3 overexpression conferred protection. These effects were accompanied by significant alterations in ferroptosis-related markers, including GPX4, FTH1, ACSL4, and 4-HNE. Moreover, the Ferroptosis inhibitor liproxstatin-1 (Lip-1) reversed the aggravated injury caused by GSTM3 downregulation. By contrast, modulation of GSTM3 expression did not significantly affect CONV-RT-induced injury in either mouse testes or GC-1 cells. ScRNA-seq analysis of GC-1R cells further suggested that radiation resistance may be associated with suppression of Ferroptosis.

Conclusion: GSTM3 alleviates FLASH-RT-induced testicular injury by modulating Ferroptosis. These findings improve our understanding of FLASH-RT-induced testicular injury and suggest potential strategies to protect against this damage.

Keywords
FLASH radiotherapy; Ferroptosis; GSTM3; Radiation-resistant cells; Testicular radiation injury.
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