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

## TL;DR

Busulfan, a chemotherapy drug, causes testicular injury by triggering ferroptosis, a type of cell death involving iron and antioxidants, which could be mitigated with specific inhibitors.

## Contribution

This study identifies ferroptosis as a novel mechanism of busulfan-induced testicular injury and suggests potential therapeutic strategies.

## Key findings

- Busulfan activates ferroptosis in testicular cells, leading to mitochondrial dysfunction and germ cell loss.
- Ferroptosis inhibitors like ZnPP partially reduce busulfan-induced cell death in vitro and in vivo.
- Iron metabolism and the xCT/GPX4 pathway are central to busulfan-induced testicular injury.

## Abstract

Busulfan, a chemotherapy drug with known reproductive toxicity, was investigated for its potential role in triggering testicular injury through ferroptosis, an iron-dependent cell death pathway. This study examined the effects of busulfan on testicular function, ferroptosis-related biomarkers (e.g., GPX4, lipid peroxidation), and spermatogenesis in both in vitro (germ cell lines) and in vivo (mouse model) systems. Results demonstrated that busulfan significantly activated the ferroptotic pathway, leading to mitochondrial dysfunction, reduced antioxidant defenses, and germ cell loss. While short-term interventions with ferroptosis inhibitors partially alleviated damage, long-term reproductive outcomes remained compromised. These findings highlight ferroptosis as a key mechanism underlying busulfan-induced testicular injury, suggesting targeted inhibition of this pathway as a potential strategy to mitigate chemotherapy-associated male infertility. Further studies are needed to optimize therapeutic approaches and assess clinical applicability.

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.