Chlorpyrifos Acts as a Positive Modulator and an Agonist of N-Methyl-d-Aspartate (NMDA) Receptors: A Novel Mechanism of Chlorpyrifos-Induced Neurotoxicity

Abstract

Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide. Long-term exposure to low levels of CPF is associated with neurodevelopmental and neurodegenerative disorders. The mechanisms leading to these effects are still not fully understood. Normal NMDA receptor (NMDAR) function is essential for neuronal development and higher brain functionality, while its inappropriate stimulation results in neurological deficits. Thus, the current study aimed to investigate the role of NMDARs in CPF-induced neurotoxicity. We show that NMDARs mediate CPF-induced excitotoxicity in differentiated human fetal cortical neuronal ReNcell CX stem cells. In addition, by using two-electrode voltage clamp electrophysiology of Xenopus oocytes expressing NMDARs, we show CPF potentiation of both GluN1-1a/GluN2A (EC₅₀ ≈ 40 nM) and GluN1-1a/GluN2B (EC₅₀ ≈ 55 nM) receptors, as well as reductions (approximately halved) in the NMDA EC₅₀s and direct activation by 10 μM CPF of both receptor types. In silico molecular docking validated CPF's association with NMDARs through relatively high affinity binding (-8.82 kcal/mol) to a modulator site at the GluN1-GluN2A interface of the ligand-binding domains.

Keywords: NMDA receptors; Xenopus oocytes; chlorpyrifos; neurotoxicity; organophosphate; stem cells; two-electrode voltage clamp.

Figure 1

CPF-induced apoptosis and cell death of 4-week differentiated ReNcell CX cells. Cells were treated with 14 μM CPF, with or without 25 μM IFN, for 24 h and the percentage of live, dead, and apoptotic cells quantified using a three-color fluorescence assay. (A) Sample cell treatment showing live (evenly stained green with FDA), dead (stained red with PI), and apoptotic (mixture of FDA and PI) cells, highlighted with arrows. Scale bar is 20 µm. (B) Data points are means ± SEMs from two independent experiments in which triplicate wells were assessed with 6 captured images in each well from the control and treated cells. For marked significance: * p < 0.05 and *** p < 0.001 based on a Bonferroni post hoc correction test for significant differences from control.

Figure 2

CPF potentiation of NMDA/glycine (10 μM/10 μM) responses in GluN1-1a/GluN2A and GluN1-1a/GluN2B-containing NMDARs. Sample TEVC recordings for CPF potentiation of current mediated by GluN1-1a/GluN2A (A) or GluN1-1a/GluN2B (B) at −75 mV. (C): Concentration–potentiation curves for CPF potentiated current mediated by GluN1-1a/GluN2A (n = 5) or GluN1-1a/GluN2B (n = 6). Percentage of potentiation (mean ± SEM) values were plotted against Log₁₀ CPF concentration and fitted with Equation (1).

Figure 3

TEVC recordings showing antagonism of CPF-induced potentiation of NMDA/Gly-evoked currents in Xenopus oocytes expressing recombinant NMDARs. The 1 μM CPF potentiated NMDA/Gly-evoked current when co-applied with 10 μM NMDA and 10 μM Gly, was antagonized by co-application of 10 μM MK-801 or 100 μM IFN to Xenopus oocytes expressing GluN1-1a/GluN2A (A) or GluN1-1a/GluN2B (B), respectively. Recordings with or without antagonist were made from the same oocytes at −75 mV.

Figure 4

CPF reduced the NMDA EC₅₀ and increased its maximum response. Concentration–response curves for NMDA-evoked (0.1 to 1000 µM; all with 10 μM Gly) current mediated by GluN1-1a/GluN2A (n = 5) (A) or GluN1-1a/GluN2B (n = 6) (B) in the absence and presence of 10 μM CPF. All data were expressed as a percentage of the response to 1 mM NMDA (mean ± SEM), plotted and fitted with Equation (1) to give EC₅₀ and maximum response values.

Figure 5

Effect of CPF alone or co-application of CPF with either NMDA or glycine in Xenopus oocytes expressing NMDARs. (A,B): TEVC recordings (at −75 mV) for direct application of 10 μM CPF to Xenopus oocytes expressing GluN1-1a/GluN2A (A) or GluN1-1a/GluN2B (B) showing elicited currents in both cases. (C,D): TEVC recordings for application of either 10 μM Gly ± 1 μM CPF or 100 μM NMDA ± 10 μM in Xenopus oocytes expressing GluN1-1a/GluN2A (C) or GluN1-1a/GluN2B (D), respectively. (E,F): Currents were significantly enhanced for glycine at GluN1-1a/GluN2A (n = 3) (E) and for NMDA at GluN1-1a/GluN2B (n = 5) (F). Data shown are means ± SEM of the peak current response. Statistical analysis was performed using an unpaired Student’s t-test (two-tailed). For marked significance: * indicates changes that were significantly different from NMDA- or glycine-only-evoked responses with p ˂ 0.05.

Figure 6

Three-dimensional (A,B) and two-dimensional (C) poses showing interaction of CPF with the GluN1/GluN2A LBD interface. CPF forms a hydrogen bond (dashed line) with Arg248(755) and hydrophobic interactions with Ile128(519), Pro141(532), Ser249(756), and Gly250(757) from the GluN1 subunit LBD and hydrophobic interactions with Val128(526), Pro129(527), Phe130(528), Val131(529), Glu132(530), Leu263(780), and Val266(783) from the GluN2A subunit LBD (numbers in parentheses are residue numbers in the full length subunits).