Prioritizing phthalate esters (PAEs) using experimental in vitro/vivo toxicity assays and computational in silico approaches

Phthalate esters (PAEs) pose prominent ecological risks owing to their multiplex toxicity potentials and ubiquitous detection in the environment. Therefore, this study aims to prioritize the individual and mixtures of six PAEs based on their toxicological implications using in vitro and vivo models exposed at environmentally relevant concentrations. Results were further confirmed using in silico Combination index (CI) and Independent action (IA), and molecular docking models. Among PAEs, DEHP revealed prominent in vitro/vivo toxicity followed by DEP, DBP, and DMP. Importantly, binary mixtures particularly C2-C6 and C11-C15 exhibited greater developmental toxicity, apoptosis, and perturbed the HPG pathway. The CI and IA models forecasted antagonistic and additive effects at Fa = 0.5 and Fa = 0.9 using in vitro Acinetobacter sp. Tox2. Conversely, in zebrafish, the IA model predicted mixture effects in the following order: additive > synergistic > antagonistic on the regulation of the HPG pathway, which was consistent with experimental results from Acridine Orange (AO) staining and apoptosis gene expression. Molecular docking for estrogen receptors (ERα, ERβ) revealed the highest binding energy scores for DEHP, compared to other PAEs. In short, our findings confirm that individual and mixtures of PAEs behave as xenoestrogens in the freshwater ecosystem with DEHP as a priority compound.