Hexabromocyclododecanes promoted autophagy through the PI3K/Akt/mTOR pathway in L02 cells

Abstract

As additive brominated flame retardants, hexabromocyclododecanes (HBCDs) are being widely used in diverse artificial materials and products, including thermal insulation building materials, housings of electronic equipment, and upholstery textiles. Toxicology studies have shown that HBCDs exposure are closely related to hepatotoxicity and liver diseases. The present study is designed to explore how HBCDs affect cell apoptosis and autophagy process in a human hepatocyte cell line (L02) and to reveal the underline molecular mechanisms. Firstly, HBCDs could elevate the apoptosis rate of L02 cells dose-dependently. Three apoptosis related proteins (apoptotic protease activating factor 1 (Apaf-1), cysteinyl aspartate specific proteinase 3 (caspase-3) and cysteinyl aspartate specific proteinase 9 (caspase-9)) were observed to be up-regulated using western blotting method. Autophagy process was also started by HBCDs in L02 cells as indicated by the increased expressions of LC3-phosphatidylethanolamine conjugate (LC3-II) and other autophagic protein markers (Beclin-1, autophagy related protein 3 (Atg3), autophagy related protein 5 (Atg5), autophagy related protein 7 (Atg7) and autophagy related protein 16L1(Atg16L1)). The results of the green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) intracellular localization and fluorescence intensity further evidenced the activation of autophagy in L02 cells after treated with HBCDs. In addition, phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway was activated in L02 cells by HBCDs, suggested by the increased expressions of related proteins. The inhibitors of PI3K (LY294002), DNA-activated protein kinase catalytic subunit (DNA-PKcs) (NU7441), Akt (MK2206), and mTOR (KU0063794) could obviously reduce the autophagic proteins prompted by HBCDs. The fluorescence intensities of GFP-LC3 transfected L02 cells were also decreased significantly after the application of these inhibitors. These results indicated that PI3K/Akt/mTOR pathway was participated in regulating autophagy process promoted by HBCDs. In above, HBCDs could induce mitochondrial-dependent apoptosis and autophagy in L02 cells, which was modulated by PI3K/Akt/mTOR pathway.