Azocyclotin Binds CDK4 to Disrupt DNA Methylation in Zebrafish Epigenetic Transgenerational Inheritance

Azocyclotin (ACT), a widely used organotin pesticide, poses environmental and health risks due to its endocrine-disrupting effects. While prior studies linked ACT to transgenerational endocrine disruption in zebrafish via DNA methylation, the underlying molecular mechanisms remained unclear. Using zebrafish ZF4 cells as an in vitro model, we elucidated the mechanism by which ACT modulates DNA methylation. ACT exposure induced a 38.03% decrease in global DNA methylation, as determined by 5-mC quantification. Integrated whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) analyses revealed that ACT dysregulated the expression of methylation-related genes, which was subsequently verified at the protein level by Western blotting. These multiomics analyses indicated that ACT disrupts the CDK4/pRb/E2F1/DNMT1 signaling axis and revealed methylation-expression coupling in endocrine-related and imprinted genes. Co-immunoprecipitation and pull-down assays demonstrated that ACT interferes with the formation of the E2F1-DNMT1-Rb-HDAC1 complex. Furthermore, molecular docking and surface plasmon resonance assays confirmed direct binding of ACT to cyclin-dependent kinase 4 (CDK4), a finding validated using CDK4-overexpressing cell lines. These findings establish CDK4 as the primary molecular target mediating ACT's epigenetic effects. This discovery provides crucial mechanistic insights into the toxicity of ACT in aquatic organisms and its potential environmental risks.