Japan Food Research Laboratories, Osaka Saito Laboratory, 7-4-41 Saitoasagi, Ibaraki-shi, Osaka, 567-0085, Japan; Osaka Prefecture University, Department of Biological Science, Graduate School of Science, 1-2 Gakuen-cho, Sakai-shi, Osaka, 599-8570, Japan; Osaka Metropolitan University, Department of Biological Chemistry, Graduate School of Science, 1-2 Gakuen-cho, Sakai-shi, Osaka, 599-8570, Japan.
Osaka Prefecture University, Department of Biological Science, Graduate School of Science, 1-2 Gakuen-cho, Sakai-shi, Osaka, 599-8570, Japan.
Toxicon. 2023 Jul;230:107161. doi: 10.1016/j.toxicon.2023.107161. Epub 2023 May 16.
Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.
雪卡鱼中毒（CFP）是一种食源性疾病，全球每年影响超过 50000 人。它是由食用积累雪卡毒素（CTXs）的海洋无脊椎动物和鱼类引起的。最近，CFP 对人类健康、当地经济和渔业资源的风险增加了；因此，迫切需要检测方法。用于检测鱼类中雪卡毒素的功能测定法包括受体结合（RBA）和神经母细胞瘤细胞测定法（N2a 测定法），这两种方法都可以检测所有 CTX 同系物。在这项研究中，我们使这些测定法更容易使用。对于 RBA，我们开发了一种使用新型近红外荧光配体 PREX710-BTX 的测定法，以节省宝贵的 CTXs。在 N2a 测定法中，我们开发了一种 1 天测定法，其检测性能与传统的 2 天测定法相同。此外，在这些测定法中，我们首次使用通过定量 NMR 确定的来自太平洋的校准 CTX 标准来比较同系物的相对效力，这与以前的研究有很大的不同。在 RBA 中，同系物之间的结合亲和力几乎没有差异，表明 CTXs 的侧链、立体化学和骨架结构的差异不会影响结合亲和力。然而，这一结果与基于急性毒性的小鼠毒力等效因子（TEFs）不相关。相比之下，N2a 测定法与基于急性毒性的小鼠 TEFs 相关性较好，CTX3C 除外。这些使用校准毒素标准获得的发现为使用功能测定法评估 CTXs 的总毒性提供了重要的见解。
