Abstract:
Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.
摘要:
Ciguatoxins(CTXs)是负责雪卡中毒(CP)的神经毒素,每年影响全球超过50,000人。预防CP的分析方法的发展是一个紧迫的全球问题,N2a测定法是检测CTX最有前途的方法之一。CTX是剧毒的,并且已经提出了在鱼中0.01μgCTX1B当量(eq)/kg的作用水平。期望进一步提高N2a测定中CTX的检测灵敏度以可靠地检测这样的低浓度。电压门控钠通道(NaV通道)的开放和电压门控钾通道(KV通道)的阻断被认为与CTX的毒性有关。因此,在这项研究中,我们开发了一种检测CTX的方法,其灵敏度高于传统的N2a检测方法,使用KV通道抑制剂作为N2a细胞的致敏试剂。向N2a细胞中添加KV通道抑制剂4-氨基吡啶和四乙基氯化铵,除了传统的致敏试剂哇巴因和维拉替丁,将N2a细胞对CTX的敏感性提高约4倍。这也是第一项在基于细胞的测定中证明KV通道对CTX毒性的影响的研究。