NETWORK TOXICOLOGY–EXPLORATION OF SWEETENER EXPOSURE AND DEPRESSION WITH IN VITRO EXPERIMENTAL VALIDATION

Objective: Sweeteners, a category of food additives are increasingly being incorporated into foods, yet their potential neurobiological safety issues remain underexplored. This study investigated the possible link between the european food safety authority (EFSA)-approved sweeteners and depression by integrating network toxicology, molecular docking, and experimental validation, with a particular focus on steviol glycoside. Methods: Sweeteners collected from EFSA were initially screened using SwissADME and ADMETlab 3.0 for toxicity profiles. The potential targets, protein–protein interactions (PPI), molecular pathways, and binding affinities of the screened compounds were further analyzed using varioussuitable in silico tools (including SwissTargetPrediction, PharmMapper, STRING, shinyGO, cytoscape, cytoHubba, molecular docking) and in vitro studies. Results: A total of 31 sweeteners were retrieved from the EFSA database and evaluated for their ADMET characteristics. Among them, 14 compounds were predicted to exhibit toxicological properties. Integration of target prediction data for these compounds with depression-related genes obtained from DisGeNET, GeneCards, Swiss Target Prediction and PharmMapper identified 225 overlapping targets, with network analysis highlighting AKT1, SRC, TP53, ALB, and ESR1 as major hub genes. Functional enrichment analysis using ShinyGO revealed significant associations with neuroactive ligand–receptor interactions, oxidative stress pathways, and mood-related signaling processes. Molecular docking analysis of the top five sweeteners indicated that steviol glycoside displayed the highest affinity and selectivity toward ESR1. Supporting these, differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy confirmed steviol-induced conformational alterations in bovine serum albumin. Additionally, biochemical assays demonstrated increased protein carbonylation (p < 0.05), elevated amadori product formation, reduced thiol content, and dose-dependent modulation of monoamine oxidase enzyme. Conclusion: These findings suggest that certain EFSA-approved sweeteners, especially steviol glycoside, may influence depression-related pathways through modulation of key molecular targets. It also induced oxidative stress and altered activity of monoamine oxidases. These results underscore the need for further mechanistic and in vivo studies to evaluate potential neurobiological risks associated with increased sweetener exposure.