New insights into mechanisms of valproic acid-induced neurodevelopmental toxicity in autism spectrum disorder: An integrative network toxicology approach combined with in vivo validation.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with growing evidence linking its etiology to chemical exposures, including valproic acid. This study employed an integrative approach combining network toxicology, molecular docking, Mendelian randomization (MR), GEO data analysis, and experimental validation to systematically elucidate the mechanisms of valproic acid (VPA)-induced neurodevelopmental toxicity in ASD. Computational toxicity predictions identified hepatotoxicity, respiratory toxicity, and pronounced blood-brain barrier penetration as key features of VPA. Network toxicology analysis predicted core targets, including epigenetic regulators (HDAC1, SIRT1), drug-metabolizing enzymes (CYP3A4, CYP2C19), and signaling mediators (RXRA, ESR1, RELA, NOS1), as pivotal in mediating VPA's neurodevelopmental toxicity. KEGG enrichment analysis highlighted alterations in neuroactive ligand-receptor interactions, CYP450 metabolism, and estrogen signaling. MR analysis only suggested a weak causal link between SIRT1 and ASD risk (OR = 1.073, p = 0.022), underscoring that VPA may contribute to ASD more through environmental perturbation than through strong genetic predisposition. Prenatal VPA-exposed rats exhibited core autistic-like behaviors (social deficits and repetitive behaviors), with neuronal degeneration in the prefrontal cortex, hippocampal dentate gyrus, and striatum. Western blotting and GEO data revealed downregulated HDAC1 and ESR1 expression, while fecal metabolomics identified 383 differentially abundant metabolites. Importantly, we establish CYP450-dependent metabolic dysregulation as a novel core mechanism in VPA-induced ASD, significantly affecting steroid hormone biosynthesis and arachidonic acid metabolism. This study underscores VPA's multi-target toxicity in ASD via epigenetic dysregulation, neuroinflammation, CYP450-dependent pathways, and gut-brain axis neurotransmitter disturbances, providing a framework for understanding chemical contributions to ASD pathogenesis.