MEHP induced mitochondrial damage by promoting ROS production in CIK cells, leading to apoptosis, autophagy, cell cycle arrest.

Although Mono (2-ethylhexyl) phthalate (MEHP) is a metabolite of Di (2-ethylhexyl) phthalate (DEHP), it has been confirmed to exhibit stronger biological toxicity than DEHP. Mitochondrial dynamic homeostasis and normal mitochondrial function regulate numerous physiological and pathological processes. However, it remains unclear whether MEHP triggers apoptosis, autophagy, and cell cycle arrest in grass carp kidney (CIK) cells by causing mitochondrial damage. Here, we established a MEHP dose-dependent exposure models in CIK cells and treated them with NAC. The results demonstrated that MEHP promoted ROS production and decreased antioxidant enzyme activities in CIK cells in a concentration-dependent manner. MEHP destroyed mitochondrial homeostasis and mitochondrial function in CIK cells, manifested by decreasing mitochondrial membrane potential (MMP), down-regulating gene expression of fusion division genes including MFN1, MFN2, CLPP, DRP1, OPA1, and MFF, and reducing OXPHOS complex enzyme protein level including COXI, COXII, COXIII, COXIV, and COXV. In addition, MEHP treatment not only can increase the level of Cyt-c, Atg12, Atg13, Atg14, Beclin1, ULK1, LC3-II, Caspase3, Caspase9, and Bax, but also can decrease the level of Bcl2, p62, CyclinB, CyclinD, and CyclinE in a concentration-dependent manner, which resulted in apoptosis, autophagy and cell cycle arrest. Furthermore, MEHP dose-dependently nduced downregulation gene expression of immunoglobulins and antimicrobial peptides (Hepcidin, β-defensin, LEAP2). However, NAC treatment could significantly reverse the above changes and alleviate CIK cells damage caused by exposure to MEHP. This study has expanded our understanding about molecular mechanisms of MEHP toxicity in aquatic animals and provided a reference for comparative medicine research.