1-NP hijacks endocrine-metabolic checkpoints and disrupts testicular steroidogenesis by suppressing the cAMP-PKA-CREB-HMGCR axis.

1-Nitropyrene (1-NP), a representative reproductive toxicant enriched in nitro-PAHs, is a known reproductive toxicant. Although our previous studies demonstrated that 1-NP impairs testosterone synthesis, its effects on other critical processes in testosterone biosynthesis, particularly cholesterol metabolism, remain unknown. Using in vivo and in vitro models, we investigated 1-NP's effects on cholesterol homeostasis and steroidogenesis. Mice were exposed to 1-NP (0, 1.25, 5 mg/kg), a mouse Leydig tumor cell line (MLTC-1) were treated with 0.1, 1 μM 1-NP along with hCG stimulation. IBMX was used for intervention experiment. Key assays included ELISA, qPCR, Western blot, filipin staining, and cholesterol/testosterone quantification. This study demonstrates that 1-NP exposure significantly depletes intracellular free cholesterol without altering total cholesterol, leading to testosterone reduction. Mechanistically, 1-NP decreases cAMP levels, impairing PKA nuclear translocation and CREB Ser133 phosphorylation, thereby downregulating the cholesterol synthesis rate-limiting enzyme HMGCR at both transcriptional and translational levels. Critically, phosphodiesterase inhibitor IBMX rescues cAMP levels, reverses HMGCR suppression, and restores free cholesterol pools and testosterone synthesis, establishing that 1-NP induces endocrine disruption via a novel cholesterol metabolic pathway. While limitations exist, this work redefines 1-NP toxicity as "metabolic sabotage" of specialized endocrine pathways, providing a framework for signal-pathway-targeted interventions against pollution-associated endocrine disruption.