Abstract
Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy. The data demonstrate that cypermethrin, α-cypermethrin, endosulfan, and chlorpyrifos concentration-dependently decreased depolarization-evoked [Ca(2+)]i, with 50% (IC50) at 78nM, 239nM, 250nM, and 899nM, respectively. Additionally, acute exposure to chlorpyrifos or endosulfan (10μM) induced a modest increase in basal [Ca(2+)]i, amounting to 68 ± 8nM and 53 ± 8nM, respectively. Imidacloprid did not disturb basal or depolarization-evoked [Ca(2+)]i at 10μM. Following exposure to binary mixtures, effects on depolarization-evoked [Ca(2+)]i were within the expected effect additivity range, whereas the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca(2+)]i in PC12 cells by inhibiting voltage-gated calcium channels (VGCCs) in vitro at concentrations comparable with human occupational exposure levels. Moreover, the effective concentrations in this study are below those for earlier described modes of action. Because inhibition of VGCCs appears to be a common and potentially additive mode of action of several classes of insecticides, this target should be considered in neurotoxicity risk assessment studies.
Highlights
ABBREVIATIONS [Ca2+ ]i CI EI inhibition of the depolarization-evoked [Ca2+]i of 20% (IC20) IC50 MOE MRL intracellular calcium concentration confidence intervals expected inhibition concentration that induces an inhibitory effect of 20% concentration that induces an inhibitory effect of 50% margin of exposure maximum residue level nicotinic acetylcholine receptors (nAChR) NOEC OP TR voltage-gated calcium channels (VGCCs) voltage-gated sodium channels (VGSCs) nicotinic acetylcholine receptor no-observed-effect concentration organophosphate treatment ratio voltage-gated calcium channel voltage-gated sodium channel
Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (␣-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca2+]i)
Following exposure to binary mixtures, effects on depolarization-evoked [Ca2+]i were within the expected effect additivity range, whereas the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca2+]i in PC12 cells by inhibiting voltage-gated calcium channels (VGCCs) in vitro at concentrations comparable with human occupational exposure levels
Summary
ABBREVIATIONS [Ca2+ ]i CI EI IC20 IC50 MOE MRL intracellular calcium concentration confidence intervals expected inhibition concentration that induces an inhibitory effect of 20% concentration that induces an inhibitory effect of 50% margin of exposure maximum residue level nAChR NOEC OP TR VGCC VGSC nicotinic acetylcholine receptor no-observed-effect concentration organophosphate treatment ratio voltage-gated calcium channel voltage-gated sodium channel. Pyrethroids induce neuronal overexcitation by delaying the inactivation of voltage-gated sodium channels (VGSCs; Soderlund, 2012). They are rapidly metabolized in humans (e.g., Leng et al, 2006) and mammals (Wolansky and Harrill, 2008). Acute oral exposure to cypermethrin (and other pyrethroids) can affect behavior in mice and rats (Wolansky and Harrill, 2008) and was shown in vitro to have several targets, including VGSCs (Meacham et al, 2008), potassium channels (YuTao et al, 2009), and ATPase (Kakko et al, 2004). In vitro research demonstrated that endosulfan exposure affects a number of neurotoxicological targets and endpoints, such as caspase-3, NFB, formation of reactive oxygen species as well as GABAA and glycine-gated chlorine channels (Jia and Misra, 2007; Vale et al, 2003)
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