Dibutyl phthalate affects insulin synthesis and secretion by regulating the mitochondrial apoptotic pathway and oxidative stress in rat insulinoma cells

Abstract

Dibutyl phthalate (DBP) is a typical phthalate (PAEs). The environmental health risks of DBP have gradually attracted attention due to the common use in the production of plastics, cosmetics and skin care products. DBP was associated with diabetes, but its mechanism is not clear. In this study, an in vitro culture system of rat insulinoma (INS-1) cells was established to explore the effect of DBP on insulin synthesis and secretion and the potential mechanisms. INS-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and treated with 15, 30, 60 and 120 μmol/L of DBP and dimethyl sulfoxide (vehicle, < 0.1%) for 24 h. The contents of insulin in the intracellular fluid and the extracellular fluid of the cells were measured. The results showed that insulin synthesis and secretion in INS-1 cells were significantly decreased in 120 μmol/L DBP group. The apoptosis rate and mitochondrial membrane potential of INS-1 cells were measured by flow cytometry with annexin V-FITC conjugate and PI, and JC-1, respectively. The results showed that DBP caused an increase in the apoptosis rate and a significant decrease in the mitochondrial membrane potential in INS-1 cells in 60 μmol/L and 120 μmol/L DBP group. The results of western blot showed that the expression of Bax/Bcl-2, caspase-3, caspase-9 and Cyt-C were significantly increased. Meanwhile, the level of oxidative stress in INS-1 cells was detected by fluorescent probes DCFH-DA and western blot. With the increase of DBP exposure, the oxidative stress levels (MDA, GSH/GSSG) were increased; and the antioxidant index (SOD) levels were decreased. Our experimental results provide reliable evidence that DBP induced apoptosis and functional impairment in INS-1 cells through the mitochondrial apoptotic pathway and oxidative stress. Therefore, we hypothesized that interference with these two pathways could be considered in the development of preventive protection measures.

Keywords: Cell toxicity; INS-1 cells; Mitochondrial apoptotic pathway; Phthalates.