GSK3 beta signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells

Fipronil (FPN) is a phenylpyrazole insecticide acted on insect gamma-aminobutyric acid (GABA) receptors. Although action of FPN is restricted on insect neuronal or muscular transmitter system, a few studies have assessed the effects of this neurotoxicant on neuronal cell death. To determine the mechanisms underlying FPN-induced neuronal cell death, we investigated whether reactive oxygen species (ROS) plays a role in FPN-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. FPN was cytotoxic to these cells and its cytotoxicity showed a concentration-dependent manner. Additionally, FPN treatment significantly decreased the tyrosine hydroxylase (TH) expression without change of glutamic acid decarboxylase 65 (GAD65) expression. FPN-induced dopaminergic cell death involved in increase of ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FPN treatment, dopamine (DA) levels decreased significantly in both cell and culture media, and oxidative effects of DA were blocked by NAC pretreatment. We showed that cell death in response to FPN was due to apoptosis since FPN increased cytochrome c release into the cytosol and activated caspase-3. It also led to nuclear accumulation of p53 and reduced the level of Bcl-2 protein in a concentration-dependent manner. Additionally, FPN altered the level of Akt/glycogen synthase kinase-3 (GSK3 beta) phosphorylation. FPN reduced the Akt phosphorylation on Ser473, and in parallel with the inactivation of Akt, phosphorylation of GSK3 beta on Ser9 which inactivates GSK3 beta, decreased after treatment with FPN. Furthermore, inhibition of the GSK3 beta signal protected the cell against FPN-induced cell death. These results suggest that regulation of GSK3 beta activity may control the apoptosis induced by FPN-induced oxidative stress associated with neuronal cell death. Crown Copyright (C) 2011 Published by Elsevier Ireland Ltd. All rights reserved.This work was supported by a grant from "Research Program for Agriculture Science & Technology Development (Agenda No. 4-12-27, 2009)" Rural Development Administration, Republic of Korea, and by a grant from the Korea Science and Engineering Foundation (2010-0029506) through the MRC for Regulation of Stem Cell Behaviors at Hanyang University, College of Medicine, Republic of Korea