Acute disturbance of calcium homeostasis in PC12 cells as a novel mechanism of action for (sub)micromolar concentrations of organophosphate insecticides.
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 (20min) 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 (10μM) induced a modest increase in basal [Ca(2+)]i. More importantly, the tested OPs concentration-dependently inhibited depolarization-evoked [Ca(2+)]i. Chlorpyrifos already induced a ∼30% inhibition at 0.1μM and a 100% inhibition at 10μM (IC50=0.43μM), whereas parathion-ethyl inhibited the depolarization-evoked [Ca(2+)]i increase with ∼70% at 10μM. 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.
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.
Marieke
M
Meijer
Institute for Environmental Studies (IVM), VU University Amsterdam, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands.
Timo
T
Hamers
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. Electronic address: r.westerink@uu.nl.
Remco H S
RHS
Westerink
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005071:Evoked Potentials / Q000187:drug effects; D016257:Fura-2 / Q000031:analogs & derivatives; D006706:Homeostasis / Q000187:drug effects*; D007306:Insecticides / Q000494:pharmacology*; D010755:Organophosphates / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats
D007306:Insecticides; D010755:Organophosphates; C049925:fura-2-am; D000110:Acetylcholinesterase; D002118:Calcium; D016257:Fura-2
Carbaryl; In vitro neurotoxicology; Organophosphates; Pesticide mixture toxicity; Single-cell fluorescent calcium imaging; Voltage-gated calcium channels