Abstract

Triazole fungicides are ubiquitous in agricultural practices, yet the multifaceted mechanisms underlying their hepatotoxicity remain inadequately defined. In this study, we employed an integrated multi-omics framework, incorporating network toxicology, transcriptomics, untargeted metabolomics, and molecular docking, to investigate the effects of flusilazole (10 μM) on AML12 mouse hepatocytes. Molecular docking established a robust mechanistic nexus between flusilazole and Cytochrome P450 (CYP) enzymes, revealing high binding affinities for CYP51, CYP1A1, and CYP2D22. These interactions correlated with a profound systemic disruption of lipid homeostasis, specifically the blockade of steroid hormone biosynthesis and the perturbation of sphingolipid metabolism. Integrated O2PLS and pathway analysis identified concordant transcriptomic and metabolomic alterations in the tryptophan-NAD + and pentose phosphate pathways. These findings suggest a hypothesized metabolic cascade wherein flusilazole-induced enzymatic inhibition shunts tryptophan flux away from de novo NAD + biosynthesis, potentially triggering a bioenergetic crisis. This is further evidenced by a compensatory shift toward the pentose phosphate pathway and the activation of IL-17/TNF stress-response signaling. Comparative analysis further distinguishes flusilazole through its specific suppression of circadian rhythm and DNA replication signatures. Collectively, our results provide a holistic and data-anchored perspective on flusilazole-induced hepatotoxicity, highlighting the interplay between enzymatic blockade, metabolic reprogramming, and chronobiological desynchronization as critical drivers of triazole-mediated hepatic injury. Synopsis Flusilazole, a widely used triazole fungicide, disrupts endocrine balance and amplifies mixture toxicity risks through conserved biological targets, threatening ecosystem and human health.