German Federal Institute for Risk Assessment, Department Safety in the Food Chain, National Reference Laboratory of Marine Biotoxins, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany - www.bfr.bund.de; Department of Pharmacy, School of Medicine and Surgery, University of Napoli Federico II, Via D. Montesano 49, 80131, Napoli, Italy.
German Federal Institute for Risk Assessment, Department Safety in the Food Chain, National Reference Laboratory of Marine Biotoxins, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany - www.bfr.bund.de.
Harmful Algae. 2021 Mar;103:101994. doi: 10.1016/j.hal.2021.101994. Epub 2021 Feb 2.
Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices; requiring sensitive, reliable, and robust methods for their detection. The mouse neuroblastoma (Neuro-2a) cytotoxicity assay (N2a-assay) is a sensitive, high-throughput, in vitro method effective for detecting sodium channel-specific marine biotoxins. The N2a-assay can be conducted to distinguish between specific effects on voltage-gated sodium (Na) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target Na. The sensitivity and specificity of the assay to compounds activating the Na are achieved through the addition of the pharmaceuticals ouabain (O) and veratridine (V). However, these compounds can be toxic to Neuro-2a cells and their application at insufficient or excessive concentrations can reduce the effectiveness of this assay for marine toxin detection. Therefore, during growth incubation, Neuro-2a cells were exposed to O and V, and surviving cells exhibiting a lower sensitivity to O and V (OV-LS) were propagated. OV-LS Neuro-2a cells were selected for 60-80% survival when exposed to 0.22/0.022 mM O/V during the cytotoxicity assay. At these conditions, OV-LS N2a cells demonstrated a 3.5-fold higher survival rate 71% ± 7.9 SD (n = 232), and lower sensitivity to O/V, compared to the original Neuro-2a cells 20% ± 9.0 SD (n = 16). Additionally, OV-LS N2a cells were 1.3-2.6-fold more sensitive for detecting CTX3C 1.35 pg/ml, CTX1B 2.06 pg/ml, and PbTx-3 3.04 ng/ml compared to Neuro-2a cells using 0.1/0.01 mM O/V. Detection of CTX3C in a complex fish matrix using OV-LS cells was 0.0048 pg CTX3C/mg fish tissue equivalent. This work shows the potential for a significant improvement in sensitivity for CTX3C, CTX1B, and PbTx-3 using the OV-LS N2a-assay.
海产食品中积累的海洋生物毒素对人类健康构成威胁。这些毒素通常在微量的情况下存在，且包含在复杂的基质中；因此需要敏感、可靠和强大的方法来进行检测。鼠神经母细胞瘤（Neuro-2a）细胞毒性测定（N2a 测定法）是一种灵敏、高通量的体外方法，可有效检测钠通道特异性海洋生物毒素。N2a 测定法可用于区分毒素对电压门控钠（Na）通道的特定影响，这些毒素可激活（例如，雪卡毒素（CTXs）、短裸甲藻毒素（PbTxs））或阻断（例如，河豚毒素、石房蛤毒素）目标 Na。通过添加药物哇巴因（O）和藜芦碱（V），该测定法对激活 Na 的化合物具有敏感性和特异性。然而，这些化合物对 Neuro-2a 细胞有毒性，其应用浓度不足或过高会降低该测定法对海洋毒素检测的有效性。因此，在生长孵育期间，将 O 和 V 暴露于 Neuro-2a 细胞，并对表现出对 O 和 V 较低敏感性（OV-LS）的存活细胞进行繁殖。当在细胞毒性测定中暴露于 0.22/0.022 mM O/V 时，OV-LS Neuro-2a 细胞的选择存活率为 60-80%。在这些条件下，与原始 Neuro-2a 细胞 20%±9.0 SD（n=16）相比，OV-LS N2a 细胞的存活率提高了 3.5 倍，达到 71%±7.9 SD（n=232），并且对 O/V 的敏感性降低。此外，与 Neuro-2a 细胞相比，OV-LS N2a 细胞对检测 CTX3C（1.35 pg/ml）、CTX1B（2.06 pg/ml）和 PbTx-3（3.04 ng/ml）的灵敏度提高了 1.3-2.6 倍，使用 0.1/0.01 mM O/V。使用 OV-LS 细胞在复杂的鱼基质中检测 CTX3C 的检测限为 0.0048 pg CTX3C/mg 鱼组织当量。这项工作表明，使用 OV-LS N2a 测定法，CTX3C、CTX1B 和 PbTx-3 的检测灵敏度有显著提高的潜力。
