The herbicide 2,4-dichlorophenoxyacetic acid induces pancreatic β-cell death via oxidative stress-activated AMPKα signal downstream-regulated apoptotic pathway

Abstract

2,4-Dichlorophenoxyacetic acid (2,4-D) is one of commonly and widely used organic herbicides in agriculture. It has been reported that 2,4-D can induce adverse effects in mammalian cells. Epidemiological and animal studies have indicated that exposure to 2,4-D is associated with poorer glycemic control and impaired pancreatic β-cell function. However, limited information is available on 2,4-D-induced toxicological effects in β-cells, with the underlying toxicological mechanisms remains unclear. Herein, our results showed that 2,4-D exposure (30-500 μg/mL) significantly reduced cell viability, induced mitochondria dysfunction (including the mitochondrial membrane potential (MMP) loss, the increase in cytosolic cytochrome c release, and the change in Bcl-2 and Bax protein expression), and triggered apoptotic events (including the increased population of apoptotic cells, caspase-3 activity, and caspase-3/-7 and PAPR activation) in RIN-m5F β-cells, accompanied with insulin secretion inhibition. Exposure of cells to 2,4-D could also evoke JNK, ERK1/2, p38, and AMP-activated protein kinase (AMPK)α activation as well as reactive oxygen species (ROS) generation. Pretreatment of cells with compound C (an AMPK inhibitor) and the antioxidantN-acetylcysteine (NAC), but not that SP600125/PD98059/SB203580 (the inhibitors of JNK/ERK/p38, respectively), obviously attenuated the 2,4-D-triggered AMPKα phosphorylation, MMP loss, apoptotic events, and insulin secretion dysfunction,as similar effects with the transfection with AMPKα1-specific siRNA. Of note, buffering the ROS production with NAC obviously prevented the 2,4-D-induced ROS generation as well as AMPKα activation, but the either compound C and AMPKα1-specific siRNA transfection could not effectively reduce 2,4-D-induced ROS generation. Collectively, these findings indicate that the induction of oxidative stress-activated AMPKα signaling is a crucial mechanism underlying 2,4-D-triggered mitochondria-dependent apoptosis, ultimately leading to β-cell death.

Keywords: 2,4-dichlorophenoxyacetic acid; AMPKα; Apoptosis; Mitochondria; Pancreatic β-cells; ROS.