Department of Chemistry, Chung Yuan Christian University, Chung-Li, Taoyuan City 320314, Taiwan.
Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taoyuan City 3020314, Taiwan.
Toxins (Basel). 2021 Apr 3;13(4):259. doi: 10.3390/toxins13040259.
Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.
槟榔碱是一种烟酸基生物碱，存在于槟榔果中。除了作为毒蕈碱激动剂的功能外，槟榔碱还表现出多种不良反应，如诱导生长迟缓，并导致动物胚胎（包括斑马鱼、鸡和小鼠）发育缺陷。在这项研究中，我们旨在研究水槟榔碱暴露对斑马鱼幼虫运动活动的潜在不良影响，并研究槟榔碱对斑马鱼行为影响的可能机制。进行了斑马鱼行为分析，以及使用毒蕈碱型乙酰胆碱受体拮抗剂的分子对接和拮抗剂共暴露实验。将受精后 96 小时（hpf）的斑马鱼幼虫暴露于不同浓度（0.001、0.01、0.1 和 1 ppm）的槟榔碱中 30 分钟和 24 小时，分别以找出不同时间暴露下槟榔碱的作用。在 120 hpf 时测量和量化运动活动。结果表明，槟榔碱即使在非常低的浓度下也会引起斑马鱼幼虫的运动过度活跃。为了进行机制研究，我们进行了基于结构的分子对接模拟和拮抗剂共暴露实验，以探索槟榔碱与斑马鱼内源性毒蕈碱型乙酰胆碱受体（mAChR）的 8 个亚型（M1a、M2a、M2b、M3a、M3b、M4a、M5a 和 M5b）之间的潜在相互作用。预测槟榔碱与大多数亚型表现出很强的结合亲和力。我们还发现，用 M1 到 M4 mAChR 拮抗剂共孵育可以挽救槟榔碱引发的运动过度活跃表型。总之，通过药理学方法，我们证明槟榔碱作为一种高活性刺激化合物，在斑马鱼中发挥作用，通过多种毒蕈碱型乙酰胆碱受体介导。
