Improving in vitro ciguatoxin and brevetoxin detection: selecting neuroblastoma (Neuro-2a) cells with lower sensitivity to ouabain and veratridine (OV-LS)

Christopher R Loeffler; Dorina Bodi; Luciana Tartaglione; Carmela Dell'Aversano; Angelika Preiss-Weigert

doi:10.1016/j.hal.2021.101994

Improving in vitro ciguatoxin and brevetoxin detection: selecting neuroblastoma (Neuro-2a) cells with lower sensitivity to ouabain and veratridine (OV-LS)

Harmful Algae. 2021 Mar:103:101994. doi: 10.1016/j.hal.2021.101994. Epub 2021 Feb 2.

Authors

Christopher R Loeffler¹, Dorina Bodi², Luciana Tartaglione³, Carmela Dell'Aversano³, Angelika Preiss-Weigert²

Affiliations

¹ 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. Electronic address: Christopher.Loeffler@bfr.bund.de.
² 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.
³ Department of Pharmacy, School of Medicine and Surgery, University of Napoli Federico II, Via D. Montesano 49, 80131, Napoli, Italy; CoNISMa - Italian Interuniversity Consortium on Marine Sciences, Piazzale Flaminio 9, 00196 Rome, Italy.

PMID: 33980434
DOI: 10.1016/j.hal.2021.101994

Abstract

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_V) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target Na_V. The sensitivity and specificity of the assay to compounds activating the Na_V 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.

Keywords: Ciguatera poisoning; Ciguatoxin, brevetoxin; Marine biotoxin; Marine toxin detection; Neuro-2a cell-based assay; Neurotoxic shellfish poisoning.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line, Tumor
Ciguatoxins* / toxicity
Marine Toxins / toxicity
Neuroblastoma*
Ouabain
Oxocins
Veratridine

Substances

Marine Toxins
Oxocins
Ciguatoxins
Ouabain
Veratridine
brevetoxin