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.

啶虫脒 (ACE) 和吡虫啉 (IMI) 是广泛使用的新烟碱类杀虫剂 (NNI)，对人类乙酰胆碱烟碱受体具有功能活性，因此具有假定的毒性作用。本研究的目的是评估 NNI 和 α7-nAChR 之间的相互作用，因为该受体使细胞内 Ca2+ ([Ca2+]i) 保持最佳状态，以实现足够的神经元功能。通过分子对接鉴定了 NNI 与人类 α-7 nAChR 的冷冻电镜结构之间可能存在的相互作用。此外，还分析了神经母细胞瘤 SH-SY5Y 细胞中的 NNI 效应，因为它们天然表达 α-7 nAChR。功能研究包括未分化 SH-SY-5Y 细胞中的增殖/细胞毒性作用（MTT 测试）以及负载 Fluo-4 AM 的视黄酸分化 SH-SY-5Y 细胞中 [Ca2+]i 瞬变的间接测量。对接分析表明，IMI 和 ACE 的结合发生在与特异性 α-7 nAChR 激动剂 EVP-6124 相同的芳香笼中。IMI表现出比ACE更好的对接强度。根据 MTT 测定，低剂量 (10–50 µM) IMI 比 ACE 更能刺激神经母细胞瘤细胞增殖。在较高剂量 (250–500 µM) 下，IMI 也优于 ACE，并且由于分化的 SH-SY5Y 神经元中的 [Ca2+]i 动员，剂量依赖性地引发更突然的荧光变化。事实上，只有 IMI 减弱了尼古丁引起的细胞内荧光刺激（即尼古丁交叉脱敏）。总之，与 ACE 相比，IMI 表现出卓越的对接强度和更强大的细胞反应，这可能与 α-7nAChR 的更强活性有关。通过与 α-7nAChR 的相互作用，IMI 将证明其对人类具有较高的神经毒性潜力。需要更多的研究来调查 IMI 对神经母细胞瘤细胞的增殖作用。