Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection
The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O)...
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| Format: | Article |
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MDPI AG
2023-11-01
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| Series: | Marine Drugs |
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| Online Access: | https://www.mdpi.com/1660-3397/21/11/590 |
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| author | Sandra Raposo-Garcia Alejandro Cao Celia Costas M. Carmen Louzao Natalia Vilariño Carmen Vale Luis M. Botana |
| author_facet | Sandra Raposo-Garcia Alejandro Cao Celia Costas M. Carmen Louzao Natalia Vilariño Carmen Vale Luis M. Botana |
| author_sort | Sandra Raposo-Garcia |
| collection | DOAJ |
| description | The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels. |
| first_indexed | 2024-03-09T16:39:17Z |
| format | Article |
| id | doaj.art-943d576f853449bb854937cd63139b79 |
| institution | Directory Open Access Journal |
| issn | 1660-3397 |
| language | English |
| last_indexed | 2024-03-09T16:39:17Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Marine Drugs |
| spelling | doaj.art-943d576f853449bb854937cd63139b792023-11-24T14:53:14ZengMDPI AGMarine Drugs1660-33972023-11-01211159010.3390/md21110590Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin DetectionSandra Raposo-Garcia0Alejandro Cao1Celia Costas2M. Carmen Louzao3Natalia Vilariño4Carmen Vale5Luis M. Botana6Departamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainDepartamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainDepartamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainDepartamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainDepartamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainDepartamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainDepartamento de Farmacología, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, SpainThe growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.https://www.mdpi.com/1660-3397/21/11/590ciguatoxinssodium channelsMTTciguatera fish poisoningtoxicityLDH |
| spellingShingle | Sandra Raposo-Garcia Alejandro Cao Celia Costas M. Carmen Louzao Natalia Vilariño Carmen Vale Luis M. Botana Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection Marine Drugs ciguatoxins sodium channels MTT ciguatera fish poisoning toxicity LDH |
| title | Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection |
| title_full | Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection |
| title_fullStr | Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection |
| title_full_unstemmed | Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection |
| title_short | Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection |
| title_sort | mouse n2a neuroblastoma assay uncertainties and comparison with alternative cell based assays for ciguatoxin detection |
| topic | ciguatoxins sodium channels MTT ciguatera fish poisoning toxicity LDH |
| url | https://www.mdpi.com/1660-3397/21/11/590 |
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