Commercial glyphosate formulations exceed active ingredient toxicity via mitochondrial ROS and transcriptomic...

Glyphosate (GLY) is among the most widely used pesticides globally. Emerging evidence suggests that GLY-based herbicides (GBHs) are more toxic than GLY alone. In this study, we used HepG2 cells expressing mitochondrial-targeted Hyper7 to monitor H₂O₂ production and performed RNA sequencing to compare transcriptomic responses to GLY and a commercial GBH formulation. GBH exposure significantly elevated mitochondrial H₂O₂ levels compared to control and GLY-treated cells. Transcriptomic analysis revealed upregulation of gene ontology (GO) terms associated with oxidative stress and response to hydrogen peroxide, alongside downregulation of antioxidant enzyme genes and reduced PRDX3 protein, indicating impaired redox homeostasis in GBH-exposed cells. GBH also induced ER stress, marked by increased expression of stress-related genes (Ern1, Ddit3) and enrichment of GO terms for the unfolded protein response. GBH treatment upregulated genes involved in autophagy and apoptosis (Sqstm1, Bbc3, Map1lc3b), suggesting progressive stress response. Additionally, in GBH-treated cells metabolic pathways were altered, with higher expression of Atf3 and Ppargc1a, and enrichment of GO terms including response to glucose and lipid metabolism regulation These results indicate that GBH triggers complex stress and metabolic changes distinct from GLY alone, highlighting the importance of assessing full formulations to better evaluate potential health risks.