PPARα Activation-Mediated Testosterone Reduction as the Key Adverse Outcome Pathway for Cd-Induced Male Infertility: A Novel Integrated Approach

Cadmium (Cd)-induced male infertility and its mechanisms have been widely studied, but systematic construction of an adverse outcome pathway (AOP) network and identification of key AOPs remained unknown. This study integrated network toxicology, single-cell sequencing, NHANES research, animal experiments, and molecular modeling to construct an AOP network and identify the key AOP for Cd-induced male infertility. We constructed an AOP network comprising 9 AOPs and confirmed that PPARα activation leading to testosterone reduction and subsequent male infertility is the key AOP. Single-cell sequencing analysis showed downregulation of testosterone synthesis genes (<i>Cyp11a1</i>) in Cd-exposed Leydig cells, whereas NHANES data showed a threshold effect between blood Cd (about 1.17 μg/L) and serum testosterone, indicating that high Cd exposure can reduce serum testosterone levels. Subnetwork analysis prioritized PPARα activation and increased the number of reactive oxygen species as potential critical MIEs. <i>In vivo</i> experiments further validated that the PPARα inhibitor (GW6471) more effectively reversed Cd-evoked sperm count reduction than the antioxidant <i>N</i>-acetylcysteine. Taken together, these results validated PPARα activation-mediated testosterone reduction as the key pathway for Cd-induced male infertility and established a novel integrative paradigm for identifying key AOPs in environmental toxicology.