Cagide Eva, Becher Paul G, Louzao M Carmen, Espiña Begoña, Vieytes Mercedes R, Jüttner Friedrich, Botana Luis M
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela , 27002 Lugo, Spain.
Chem Res Toxicol. 2014 Oct 20;27(10):1696-706. doi: 10.1021/tx500188a. Epub 2014 Oct 6.
Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of (15)N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.
哈帕林多类化合物是蓝藻目颤藻科的一大类生物活性代谢产物。来自费氏藻属的12-表-哈帕林多E异腈、12-表-哈帕林多C异腈、12-表-哈帕林多J异腈和哈帕林多L对昆虫幼虫具有急性毒性；然而，目前尚不清楚负责哈帕林多类化合物生物活性的生化靶点。我们在此描述了这四种哈帕林多同系物的电子轰击质谱；它们的结构通过核磁共振光谱得到了证实。结合所呈现的（15）N标记物种的质谱及其在气相色谱毛细管柱上的保留时间，在未来的研究中应该能够实现快速可靠的测定。这些哈帕林多类化合物的生物活性在哺乳动物细胞上进行了测试，重点关注它们对BE(2)-M17兴奋性人神经母细胞瘤细胞系的影响。应用荧光染料阿拉玛蓝监测细胞毒性，用fura-2评估胞质钙浓度的变化，用双苯磺酰草胺检测对膜电位的影响。数据表明，即使将哈帕林多类化合物孵育72小时，它们也不会影响神经母细胞瘤细胞的活力。在哈帕林多类化合物处理后，细胞中既未观察到去极化，也未观察到钙内流的启动。然而，数据提供了证据表明哈帕林多类化合物是钠通道调节性神经毒素。它们以类似于新石房蛤毒素的方式抑制藜芦碱诱导的去极化。我们的数据表明，哈帕林多类化合物应被添加到越来越多的神经毒性次生代谢产物中，例如淡水蓝藻中已知的石房蛤毒素和类毒素。作为稳定的同系物，哈帕林多类化合物可能对淡水生态系统或农业用水构成风险，因此在水质评估中应予以考虑。
