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

Abstract

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. 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-resistant GC-1 cell line (GC-1R) was established and analyzed by single-cell RNA sequencing (scRNA-seq). FLASH-RT-induced testicular injury exhibited dose- and time-dependent features. At 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. 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.