A New Insight into the Mechanism of DEHP-Induced Hepatotoxicity in Adolescent Mice: Triggering Hepatic Lipid Accumulation by Targeting GRP75-Dependent MAM Formation

Abstract

Di(2-ethylhexyl) phthalate (DEHP) is widely acknowledged as a prevalent environmental pollutant, mainly due to its extensive use in plastics. This widespread application has resulted in its presence in air, soil, water, and various food items, as well as in agricultural products and tissues of both humans and animals. With the extensive use of plastic products in daily life, the teenage group is facing an increasing risk of DEHP exposure. However, the mechanism of DEHP-induced liver injury, especially during adolescence, remain unclear. This study utilized metabolomics analysis and molecular biology approaches to explore the effects of DEHP on liver function in adolescent mice. We found that DEHP exposure caused hepatic dysfunction, histopathological abnormalities, and lipid deposition. Metabolomics analysis identified the dysregulation of lipid metabolism, especially the fatty acid pathway. Mechanistically, DEHP and its main metabolite, mono(2-ethylhexyl) phthalate (MEHP) drived the ectopic formation of mitochondrial-associated endoplasmic reticulum membranes (MAMs) by up-regulating glucose-regulated protein 75 (GRP75). Such a process is characterized by a reduced interorganellar contact distance between the endoplasmic reticulum and mitochondria as well as elevated expression of MAM-resident proteins. Notably, overexpression of GRP75 alone recapitulated the lipid accumulation phenotype. Conversely, knockdown of GRP75 attenuated MEHP-induced lipid accumulation in AML12 cells. Moreover, a combination of molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assays collectively corroborated the direct physical interaction between MEHP and GRP75. Collectively, our findings demonstrate that DEHP/MEHP disrupts the lipid metabolism regulated by MAM through a GRP75-dependent mechanism, thereby leading to liver steatosis. This study clarified the toxicological mechanisms underlying DEHP-induced hepatic dysfunction in adolescent mice, providing new insights into its health risk assessment and potential intervention strategies.