Sub‑chronic dietary exposure to environmentally relevant doses of di‑(2‑ethylhexyl) adipate (DEHA) induces hepatic steatosis in female mice via disrupting the PPARγ pathway and inducing endoplasmic reticulum stress: In vivo and in vitro evidence

Di-(2-ethylhexyl) adipate (DEHA), initially regarded as a safer alternative to Di-(2-ethylhexyl) phthalate (DEHP) phthalate plasticizer, is widely used in food packaging and consumer products. Despite its high detection levels in food and established human exposure, the health impacts of DEHA at environmentally relevant doses remain poorly understood. The main aim of this study was to investigate whether sub-chronic dietary exposure to DEHA disrupts metabolism homeostasis in female mice, and to clarify the underlying mechanism through combined in vivo and in vitro models. In this study, 27 four‑week‑old female C57BL/6 J mice with similar initial body weight were randomly assigned to four groups (control (n = 6), vehicle (n = 6), low‑dose DEHA (n = 7), and high‑dose DEHA (n = 8)), where the DEHA concentrations in the mice's diet were 0.087 and 18.2 mg/kg, respectively, for 15 weeks of sub-chronic exposure. DEHA significantly increased body weight, fat mass, fasting blood glucose, serum cholesterol and induced hepatic steatosis, without affecting food intake or energy expenditure. Untargeted metabolomics analysis identified a significant elevation of lipid metabolites in the liver tissues of DEHA-exposed mice. Further mechanistic studies demonstrated that DEHA upregulated the peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathways and the expression of genes associated with endoplasmic reticulum (ER) stress. In both mouse alpha mouse liver 12 (AML12) and human liver-7702 (L02) hepatocytes, DEHA directly induced dose-dependent lipid accumulation. Pharmacological inhibition of ER stress with specific inhibitor 4-phenylbutyric acid (4-PBA) attenuated DEHA-induced lipid accumulation and lipogenic gene expression. These findings demonstrate that environmental-level DEHA exposure promoted obesity and hepatic steatosis in female mice by disturbing the PPARγ signaling pathway and upregulating ER stress-related genes, highlighting a significant and previously underappreciated health risk of DEHA in females and calling for a reassessment of its safety as an endocrine-disrupting chemical.