Acute disturbance of calcium homeostasis in PC12 cells as a novel mechanism of action for (sub)micromolar concentrations of organophosphate insecticides Marieke Meijer a , Timo Hamers b , Remco H.S. Westerink a, * a Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht, The Netherlands b Institute for Environmental Studies (IVM), VU University Amsterdam, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands 1. Introduction Organophosphates (OPs) and carbamates are insecticides used worldwide in agriculture and households. These compounds are well-known neurotoxicants that exert their adverse effect via inhibition of acetylcholine esterase (AChE), thereby inhibiting the breakdown of the neurotransmitter acetylcholine (ACh), resulting in a potentially lethal overstimulation of neurotransmission. Though the inhibition of AChE by carbamates is reversible, OPs induce an irreversible inhibition of AChE (Eaton et al., 2008; Moser et al., 2010). Moreover, some OPs inhibit neuropathy target esterase (NTE; Eaton et al., 2008), which may be responsible for some of the observed neurotoxic effects following chronic OP exposure. Though OPs are primarily known as AChE inhibitors, animal studies demonstrated neurobehavioral effects of OPs at exposure levels that do not induce AChE inhibition (Carr et al., 2001; Dam et al., 2000; Qiao et al., 2003; Ricceri et al., 2006; Timofeeva et al., 2008), suggesting the involvement of additional mechanisms. Accordingly, in vitro studies demonstrated numerous effects of OPs like chlorpyrifos and parathion, including effects on neurite outgrowth (Das and Barone, 1999; Howard et al., 2005), tyrosine hydroxylase and choline acetyltransferase (Jameson et al., 2006; Monnet-Tschudi et al., 2000), muscarinic and nicotinic acetylcho- line receptors (Betancourt and Carr, 2004; Guo-Ross et al., 2007; Liu et al., 2002; Slotkin et al., 2004; Smulders et al., 2004), adenylyl cyclase (Adigun et al., 2010), DNA synthesis (Slotkin et al., 2007), gene expression and dopamine homeostasis (Aldridge et al., 2005; NeuroToxicology 43 (2014) 110–116 A R T I C L E I N F O Article history: Received 30 September 2013 Received in revised form 2 January 2014 Accepted 18 January 2014 Available online 1 February 2014 Keywords: In vitro neurotoxicology Organophosphates Carbaryl Pesticide mixture toxicity Single-cell fluorescent calcium imaging Voltage-gated calcium channels A B S T R A C T Organophosphates (OPs) and carbamates are widely used insecticides that exert their neurotoxicity via inhibition of acetylcholine esterase (AChE) and subsequent overexcitation. OPs can induce additional neurotoxic effects at concentrations below those for inhibition of AChE, indicating other mechanisms of action are also involved. Since tight regulation of the intracellular calcium concentration ([Ca 2+ ] i ) is essential for proper neuronal development and function, effects of one carbamate (carbaryl) and two OPs (chlorpyrifos, parathion-ethyl) as well as their -oxon metabolites on [Ca 2+ ] i were investigated. Effects of acute (20 min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca 2+ ] i were measured in fura-2-loaded PC12 cells using single-cell fluorescence microscopy. Acute exposure to chlorpyrifos and its metabolite chlorpyrifos-oxon (10 mM) induced a modest increase in basal [Ca 2+ ] i . More importantly, the tested OPs concentration-dependently inhibited depolarization-evoked [Ca 2+ ] i . Chlor- pyrifos already induced a 30% inhibition at 0.1 mM and a 100% inhibition at 10 mM (IC 50 = 0.43 mM), whereas parathion-ethyl inhibited the depolarization-evoked [Ca 2+ ] i increase with 70% at 10 mM. Interestingly, -oxon metabolites were more potent inhibitors of AChE, but were less potent inhibitors of depolarization-evoked [Ca 2+ ] i compared to their parent compound (chlorpyrifos-oxon) or were even without effect (paraoxon-ethyl and -methyl). Similarly, acute exposure to carbaryl had no effect on [Ca 2+ ] i . Exposure to mixtures of chlorpyrifos with its oxon-analog or with parathion-ethyl did not increase the degree of inhibition, indicating additivity does not apply. These data demonstrate that concentration- dependent inhibition of depolarization-evoked [Ca 2+ ] i is a novel mechanism of action of (sub)micromolar concentrations of OPs that could partly underlie OP-induced neurotoxicity. ß 2014 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +31 30 2533487; fax: +31 30 2535077. E-mail address: r.westerink@uu.nl (Remco H.S. Westerink). Contents lists available at ScienceDirect NeuroToxicology 0161-813X/$ – see front matter ß 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2014.01.008