A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

Abstract

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.

Monacyclinones, New Angucyclinone Metabolites Isolated from Streptomyces sp. M7_15 Associated with the Puerto Rican Sponge Scopalina ruetzleri

“…Cytotoxicity was assessed using a live cell imaged based assay that utilizes the fluorescent nuclear stain Hoechst 33342 (H-dye) (Sigma, St Louis, MO, USA) and the fluorescent microtubule stain Tubulin Tracker 488 (Life Technologies, Gaithersburg, MD, USA) [ 35 ]. For this assay, the most sensitive cell line for marine toxins was found to be the SJCRH30 cells.…”

“…In addition to being very sensitive to marine toxins, these cells are large and spread out which allows for counting efficiency for live cell imaging and allowed for easier measurement and visualization of morphological changes associated with toxin. Comparison of the half maximal effective concentration (EC 50 ) values for the traditional XTT cytotoxicity assay and the in vitro microscopy assay using the human cell line model (SJCHR30) showed no significant difference between the assays [ 35 ], therefore the in vitro cytotoxicity assay was used to determine the EC 50 values for the marine derived products in this manuscript.…”

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodology Used in Their Control: Brevetoxins

“…Similarly, a new fluorescence microscopy method involving the use of an alternative cell line to N2A cells, was developed for the assessment of cytotoxicity. This has been reported as providing potential advantages over the traditional N2A assay, but is also yet to be applied to extracts of contaminated shellfish samples [ 106 ].…”

Surface plasmon resonance and cytotoxicity assays of drug efficacies predicted computationally to inhibit p53/MDM2 interaction

“…Hormetic dose responses are well known and commonly observed in human tumor cell lines where low and high doses of drug have opposite effects and activate different signaling pathways [36]. Hormetic apoptotic responses have been reported in irradiated zebrafish embryos and in ouabain/veratridine-treated Neuro-2A cells in response to brevetoxin [37,38]. The brevetoxins are thought to act through the activation of voltage-sensitive sodium channels in cell membranes [38].…”

“…Hormetic apoptotic responses have been reported in irradiated zebrafish embryos and in ouabain/veratridine-treated Neuro-2A cells in response to brevetoxin [37,38]. The brevetoxins are thought to act through the activation of voltage-sensitive sodium channels in cell membranes [38].…”