DINCH induces intestinal toxicity through "ROS-ENO1-PA-lipid peroxidation" axis: An integrated approach combining...

Cyclohexane-1,2-dicarboxylic acid diisononyl ester (DINCH), as a "green" alternative to phthalates, has been widely used in consumer products and industrial materials. Consequently, its environmental and human exposure levels continue to rise, necessitating a systematic evaluation of its mechanism of intestinal toxicity. This study developed a strategy integrating "network toxicology-machine learning-molecular simulation-experimental validation": First, multiple databases were combined to identify potential intestinal toxicity targets of DINCH, followed by the construction of a PPI network and the identification of the core pathway "metabolic pathway" through GO/KEGG enrichment. Subsequently, ENO1 was selected by machine learning. Molecular docking and molecular dynamics simulations consistently demonstrate a interaction between DINCH and ENO1. In vitro experiments confirmed that high concentrations of DINCH significantly downregulated ENO1 expression and attenuated the H₂O₂-induced increase in ENO1 levels, leading to ROS accumulation, mitochondrial dysfunction, and lipid peroxidation. Furthermore, experiments with ENO1 gene silencing demonstrated that DINCH-induced lipid peroxidation was dependent on ENO1. Exogenous supplementation of PA significantly reversed DINCH-triggered mitochondrial damage, and lipid peroxidation. NAC and Mito-TEMPO interventions also alleviated the H₂O₂ + DINCH-induced lipid peroxidation. In summary, this study is the first to elucidate the novel molecular mechanism by which DINCH aggravates H₂O₂-induced ROS-ENO1-dependent oxidative stress, providing critical scientific evidence for revising its safety thresholds, managing exposure risks, and developing next-generation alternative plasticizers.