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Updated: Jun 10, 2026
An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity
Published on: May 12, 2020
Cytotoxic quantification of maitotoxin-like activity from the dinoflagellate Gambierdiscus toxicus
G Diogène1, A Dubreuil, J P Breittmayer
1Unité INSERM 303, BP 3, Villefranche-sur-Mer, F-06230, France.
Abstract:
Gambierdiscus toxicus is a marine dinoflagellate involved in the food-borne disease ciguatera. Its toxicity is mainly due to maitotoxin, a hydrophilic toxin, the chemical structure of which has recently been described. This toxin increases internal Ca(2+) concentration and triggers phosphoinositide breakdown. Three cytotoxic tests were set for the detection and quantification of toxicity in G. toxicus extracts, further purified by HPLC: the neutral red uptake assay (NRU), observation of morphological alterations on baby hamster kidney fibroblasts (BHK 21/C13) and the measurement of internal Ca(2+) concentration on human lymphocytes (Jurkat D). In order to evaluate cytotoxicity tests, results were compared with the mouse bioassay. A positive correlation was observed between the minimum lethal dose (MLD), estimated at 24 hr by the mouse bioassay, and the IC(50), estimated by the NRU assay, for extracts of increasing degree of purity. By the use of the NRU assay, a precise detection and quantification of toxicity in multiple extracts was possible after 8 hr. Cell blebbing was generally highest for extracts that strongly inhibited NRU. The analysis of the correlation between the two cytological tests facilitates monitoring of the progression of purification. An indication of the presence of maitotoxin activity was supported by the increase of internal Ca(2+) concentration in Jurkat D lymphocytes, which was maximal in cells exposed to the purest extracts.
Insights
Gambierdiscus toxicus, a marine dinoflagellate causing ciguatera, can be detected using cytotoxicity assays. The neutral red uptake assay shows promise for quantifying maitotoxin toxicity in G. toxicus extracts.
Area of Science:
- Marine biology
- Toxicology
- Cell biology
Background:
- Ciguatera is a food-borne disease caused by the marine dinoflagellate Gambierdiscus toxicus.
- Maitotoxin, a potent hydrophilic toxin produced by G. toxicus, is responsible for ciguatera fish poisoning.
- Understanding maitotoxin's effects and developing reliable detection methods are crucial for public health.
Purpose of the Study:
- To evaluate and compare three cytotoxicity assays for detecting and quantifying toxicity in Gambierdiscus toxicus extracts.
- To establish correlations between these assays and the traditional mouse bioassay.
- To assess the utility of these assays in monitoring the purification of maitotoxin.
Main Methods:
- Three cytotoxicity tests were employed: neutral red uptake assay (NRU), morphological alteration observation in baby hamster kidney fibroblasts (BHK 21/C13), and intracellular calcium concentration measurement in human lymphocytes (Jurkat D).
- Extracts of G. toxicus were purified using High-Performance Liquid Chromatography (HPLC).
- Results from cytotoxicity assays were compared with the mouse bioassay (minimum lethal dose - MLD).
Main Results:
- A positive correlation was found between the mouse bioassay's MLD and the NRU assay's IC(50) for purified G. toxicus extracts.
- The NRU assay enabled precise detection and quantification of toxicity in multiple extracts within 8 hours.
- Increased intracellular calcium concentration in Jurkat D lymphocytes correlated with maitotoxin activity, peaking with the purest extracts.
Conclusions:
- The neutral red uptake assay is a reliable and efficient method for detecting and quantifying maitotoxin toxicity in G. toxicus extracts.
- Cytotoxicity assays, particularly NRU, can effectively monitor the purification process of maitotoxin.
- These assays offer a viable alternative to the mouse bioassay for evaluating G. toxicus toxicity.
