Sex-Specific sRNA Signatures in Rat Liver Reveal Divergent Alterations Following Perinatal Exposure to Glyphosate and Its Mixture with 2,4-D and Dicamba

Perinatal exposure to environmental toxicants, even at regulatory relevant doses, can disrupt molecular programming during critical developmental windows, with long-term consequences for organ function and disease risk. We investigated sex-specific hepatic microRNA (miRNA) responses in Wistar rats following perinatal exposure to glyphosate at European Union (EU) acceptable daily intake (ADI) dose (0.5 mg/kg bw/day), at no-observed-adverse-effect level (NOAEL; 50 mg/kg bw/day), and mixed with 2,4-D (0.3 mg/kg bw/day) and dicamba (0.02 mg/kg bw/day), each at their ADI. Using small RNA sequencing, we identified distinct miRNA expression profiles in males and females, with the mixture inducing the most pronounced divergence (52 differentially expressed miRNAs between males and females). Functional enrichment analysis of validated miRNA targets revealed activation of apoptotic, oncogenic, and stress-related pathways in males, alongside downregulation of homeostatic and anti-fibrotic regulators. Females showed suppression of miRNAs involved in hormone signaling, development, and tissue regeneration, suggesting endocrine and adaptive disruption. Our findings highlight the importance of determining effects based on sex and sensitive developmental stages in toxicological assessment, since different regulatory programs may be involved in the response of males and females to xenobiotics. The identified miRNAs may represent early biomarkers of hepatic dysfunction following early-life herbicide exposure, supporting their utility in future risk evaluations.