Objective To investigate the reproductive toxicity effects and mechanisms of benzene exposure in nuclear factor erythroid 2-related factor 2 (Nrf2) knockout male mice.

Methods Nrf2 knockout (Nrf2^-/-) and wild-type (WT) C57BL/6 mice were exposed to benzene via an independent ventilation cage dynamic exposure system. The concentrations of benzene were 0, 3.47, and 34.75 mg/m³ at 6 hours/day, 6 days/week, for a total of 28 days. Sperm morphological analysis and testicular histopathology were employed to assess reproductive system damage, complemented by testicular transcriptome sequencing to explore mechanisms and potential signaling pathways.

Results Nrf2^-/- exacerbated benzene-induced male reproductive system damage in mice. In Nrf2^-/- mice exposed to low-dose (3.47 mg/m³) and high-dose (34.75 mg/m³) benzene, the sperm malformation rate increased by 14.46% and 28.31%, respectively, while the sperm survival rate decreased by 8.2% and 39.2%, respectively. Testicular pathological examination showed structural abnormalities in the seminiferous tubules of Nrf2^-/- mice, with reduced lumen diameters of 57.42 and 56.61 μm and decreased epithelial thicknesses of 50.62 and 37.98 μm, accompanied by vacuolar lesions and inflammatory infiltration. In the WT mice, exposure to high-dose benzene resulted in a disorganized arrangement of seminiferous tubule epithelial cells, with a significant increase in sperm malformation rate by 24.49%. All the above indicators showed statistically significant differences (P < 0.05). Transcriptomic analysis identified perturbations in drug metabolism, oxidative stress, and cancer- related pathways.

Conclusions Benzene exposure could likely induce reproductive impairment in Nrf2^-/- mice through dysregulation of drug metabolism, cancer-related, and oxidative stress pathways. Nrf2 exerts protective effects against benzene-induced reproductive toxicity, and Nrf2 knockout aggravates male reproductive system damage.