The impact of biological sex on the efficacy of enalapril, metformin and pentoxifylline in radiation-induced lung injury

Radiation-induced lung injury (RILI) remains a significant concern following thoracic radiotherapy, progressing from radiation pneumonitis to fibrovascular remodeling and fibrosis. Transforming growth factor beta 1 (TGFβ1) and vascular endothelial growth factor (VEGF) play a role in this process, but the contribution of biological sex to the progression of RILI and treatment response is not fully defined. We evaluated sex-dependent molecular and histopathological responses in localized right-lung irradiation and compared enalapril, metformin, and pentoxifylline as candidate radioprotective interventions. A total of 130 Sprague-Dawley rats were divided into Sham, RT-only, Enalapril + RT, Metformin + RT, or Pentoxifylline + RT groups according to their sex. Rats in the irradiation groups received a single dose of 15 Gy to the right lung. Treatments were initiated 1 week prior to irradiation and continued throughout the follow-up period. Animals were sacrificed at the end of weeks 6, 12, or 24 for molecular and histopathological analyses. TGFβ1 and VEGF mRNA expression was measured by qRT-PCR, and histopathology was assessed separately in the irradiated right lung and the non-irradiated contralateral left lung using semi-quantitative scoring and alveolar wall thickness measurement. Radiation produced a sex- and time-dependent injury pattern. In the RT-only group, males showed more severe peribronchial lymphoid infiltration than females ( p = 0.010). Molecular activation peaked at week 12, when males had higher TGFβ1 and VEGF expression than females ( p = 0.009 for both). By week 24, males showed more evident chronic fibrovascular injury. Enalapril showed mainly early non-significant inflammatory trends, including neutrophilic and vascular changes, particularly in female rats, without consistent statistically significant effects on later structural endpoints. Metformin significantly reduced chronic alveolar wall thickness in males at week 24 ( p = 0.003). Pentoxifylline showed the broadest treatment-related pattern, with significant reductions in chronic peribronchial lymphoid infiltration in males ( p = 0.030) and delayed contralateral neutrophil infiltration ( p = 0.013) and edema ( p = 0.038) in females. Biological sex appeared to modify both radiation-induced injury patterns and treatment responsiveness. In the RT-only condition, male rats showed greater subacute angiogenic and profibrotic activation and more evident chronic fibrovascular remodeling, indicating a more pronounced injury phenotype after localized right-lung irradiation. Treatment effects also varied by sex and lung compartment: Pentoxifylline showed the broadest treatment-related pattern, with attenuation of chronic peribronchial lymphoid infiltration in males and delayed contralateral neutrophil infiltration and edema in females; Metformin showed a selective male-specific reduction in chronic alveolar wall thickening; and Enalapril showed mainly early non-significant inflammatory trends, including neutrophilic and vascular changes, without consistent effects on later structural endpoints. These findings highlight the need for a more tailored approach in preclinical radioprotection research, in which treatment effects are evaluated according to biological sex, post-irradiation timing, lung compartment, and the dominant histopathological endpoint rather than as a uniform response across all injury components. In addition, selective contralateral lung changes should be interpreted cautiously, as they may be consistent with delayed abscopal-like or systemic bystander responses involving inflammatory and microvascular components.