A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

Marine Drugs

Jennifer R. McCall¹  Elizabeth A. Elliott² 

[1] NCW Center for Marine Science, 5600 Marvin K Moss Lane, Wilmington, NC 28409, USA;

关键词: brevetoxins;    cytotoxicity assay;    triphasic response;    hormesis;    Neuro-2A;    SJCRH30;   

DOI  :  10.3390/md12094868

来源: mdpi

【 摘 要 】

Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC₅₀ values for brevetoxin and non-linear triphasic dose response curves. Ouabain/veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC₅₀ values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.