Hepatotoxicidade induzida pelo herbicida 2,4-D e seu metabólito 2,4-DCP: uma investigação mecanística integrando toxicologia de redes e análises em peixe-zebra

Abstract

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its main environmental metabolite, 2,4-dichlorophenol (2,4-DCP), are emerging contaminants frequently detected in aquatic ecosystems. Although their hepatotoxicity in vertebrates is already recognized, the underlying molecular mechanisms remain poorly understood. Moreover, most studies employ high concentrations and acute exposures, limiting the understanding of effects under environmentally relevant conditions characterized by prolonged exposure to low concentrations. In this study, we adopted an integrated approach combining computational toxicology and in vivo experiments using zebrafish (Danio rerio) to investigate the mechanisms of liver toxicity induced by 2,4-D and 2,4-DCP, as well as their acute and chronic effects at environmentally relevant concentrations. Network toxicology and molecular docking analyses revealed potential interactions of both compounds with key proteins associated with liver toxicity, including SRC, AKT1, RXRA, HSP90AA1, and MDM2. Functional enrichment analyses (Gene Ontology and Reactome) indicated the involvement of critical signaling pathways, including nuclear receptors and PI3K/AKT, as well as biological processes associated with liver toxicity, such as oxidative stress, lipid accumulation, mitochondrial dysfunction, and cell death. In vivo assays demonstrated that environmentally relevant concentrations (3, 30, and 300 μg/L) induced acute liver toxicity in zebrafish larvae, corroborating the in silico predicted mechanismse. In adult fish, chronic exposure resulted in histopathological alterations, including lipid accumulation, cell death, and tissue disorganization, even at 30 μg/L of 2,4-D, corresponding to the maximum allowed value (MAV) for drinking water in Brazil. This study describes, for the first time, biological targets and molecular pathways associated with 2,4-D and 2,4-DCP-induced hepatotoxicity and demonstrates that environmentally relevant concentrations can disrupt essential cellular processes, leading to liver damage. These findings question the safety of the MAV of 2,4-D established by Brazilian legislation for non-target organisms and highlight the urgent need to include 2,4-DCP in environmental monitoring programs, as well as to reassess safe exposure limits for these contaminants.