Ferroptotic Pathway Activation in Spermatogonia: A Novel Mechanism of Busulfan-Induced Testicular Injury

Busulfan (BU) is a widely used chemotherapy drug that has been shown to cause reproductive functional impairment in humans and model animals. However, the precise mechanisms underlying testicular injury induced by BU exposure have not been fully elucidated. Ferroptosis is a form of programmed cell death mediated by iron-dependent lipid peroxidation. The aim of the current study was to determine whether ferroptosis was involved in BU-induced testicular injury. We demonstrated that exposure to BU led to an increase in iron content in the testes of mice. Subsequent western blotting and reverse transcription quantitative PCR, as well as staining of testicular tissue sections, confirmed that ferroptosis mediated BU-induced testicular injury. Consistent with our in vivo findings, we found that ferroptosis, including iron metabolism and the solute carrier family 7 member 11/glutathione peroxidase 4 (xCT/GPX4) signaling pathway, may play a key role in mediating BU-induced injury to GC-1 spg cells in vitro. Treatment with ferroptosis inhibitors slowed cell death caused by BU exposure. Specifically, we found that the administration of zinc protoporphyrin IX (ZnPP), a heme oxygenase 1 (HO1) inhibitor, rescued BU-induced cell death. In conclusion, our in vivo and in vitro findings confirmed that BU exposure led to testicular ferroptosis in mice via the iron intake pathway and the HO1 signaling pathway.