Molecular Docking Analysis at the Human α7-nAChR and Proliferative and Evoked-Calcium Changes in SH-SY5Y Cells by Imidacloprid and Acetamiprid Insecticides.

Acetamiprid (ACE) and Imidacloprid (IMI) are widely-used neonicotinoid insecticides (NNIs) with functional activity at human acetylcholine nicotinic receptors and, therefore, with putative toxic effects. The objective of this study was the evaluation of the interactions between NNIs and α7-nAChR, as this receptor keeps intracellular Ca2+ ([Ca2+]i) to an optimum for an adequate neuronal functioning. Possible interactions between NNIs and the cryo-EM structure of the human α-7 nAChR were identified by molecular docking. Additionally, NNI effects were analyzed in neuroblastoma SH-SY5Y cells, as they naturally express α-7 nAChRs. Functional studies included proliferative/cytotoxic effects (MTT test) in undifferentiated SH-SY-5Y cells and indirect measurements of [Ca2+]i transients in retinoic acid-differentiated SH-SY-5Y cells loaded with Fluo-4 AM. Docking analysis showed that the binding of IMI and ACE occurred at the same aromatic cage that the specific α-7 nAChR agonist EVP-6124. IMI showed a better docking strength than ACE. According to the MTT assays, low doses (10-50 µM) of IMI better than ACE stimulated neuroblastoma cell proliferation. At higher doses (250-500 µM), IMI also prevailed over ACE and dose-dependently triggered more abrupt fluorescence changes due to [Ca2+]i mobilization in differentiated SH-SY5Y neurons. Indeed, only IMI blunted nicotine-evoked intracellular fluorescence stimulation (i.e., nicotine cross-desensitization). Summarizing, IMI demonstrated a superior docking strength and more robust cellular responses compared to ACE, which were likely associated with a stronger activity at α-7nAChRs. Through the interaction with α-7nAChRs, IMI would demonstrate its high neurotoxic potential for humans. More research is needed for investigating the proliferative effects of IMI in neuroblastoma cells.

Neurocytotoxicity of imidacloprid- and acetamiprid-based comercial insecticides over the differentiation of SH-SY5Y neuroblastoma cells.

Neonicotinoids are effective insecticides with specificity for invertebrate nicotinic acetylcholine receptors. Neonicotinoids are chemically stable and tend to remain in the environment for long so concerns about their neurotoxicity in humans do nothing but increase. Herein, we evaluated the chronic toxic effects of acetamiprid- and imidacloprid-based insecticides over the differentiation of human neuroblastoma SH-SY5Y cells, which were exposed to these insecticides at a concentration range similar to that applied to crop fields (0.01-0.5 mM). Both insecticides did not have acute cytotoxic effects in both non-differentiated and in staurosporine-differentiated SH-SY5Y cells cytotoxicity as measured by the MTT and vital-dye exclusion tests. However, after a chronic (7-day) treatment, only imidacloprid dose-dependently decreased the viability of SH-SY5Y cells (F(4,39) = 43.05, P < 0.001), largely when administered-during cell differentiation (F(4,39) = 51.86, P < 0.001). A well-defined dose-response curve was constructed for imidacloprid on day 4 (R2 = 0.945, EC50 = 0.14 mM). During differentiation, either imidacloprid or acetamiprid dose-dependently caused neurite branch retraction on day 3, likely because of oxidative stress, to the extent that cells turned into spheres without neurites after 7-day treatment. Despite their apparent safety, the neurodevelopmental vulnerability of SH-SY5Y neurons to the chronic exposure to imidacloprid and to a lesser extent to acetamiprid points to a neurotoxic risk for humans.