Reporter gene assays for algal-derived toxins

Authors

  • Elizabeth R. Fairey,

    1. Marine Biotoxins Program, NOAA-National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412, USA
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  • John S. Ramsdell

    Corresponding author
    1. Marine Biotoxins Program, NOAA-National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412, USA
    • Coastal Research Branch, NOAA-National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412, USA
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  • This article is a US Government work and is in the public domain in the USA.

Abstract

We have modified the cell-based directed cytotoxicity assay for sodium channel and calcium channel active phycotoxins using a c-fos-luciferase reporter gene construct. In this report we describe the conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method. N2A mouse neuroblastoma cells, which express voltage-dependent sodium channels, were stably transfected with the reporter gene c-fos-luc, which contains the firefly luciferase gene under the transcriptional regulation of the human c-fos response element. The characteristics of the N2A reporter gene assay were determined by dose response with brevetoxin and ciguatoxin. Brevetoxin-1 and ciguatoxin-1 induced c-fos-luc with an EC50 of 4.6 and 3.0 ng ml−1, respectively. Saxitoxin caused a concentration-dependent inhibition of brevetoxin-1 induction of c-fos-luc with an EC50 of 3.5 ng ml−1. GH4C1 rat pituitary cells, which lack voltage-dependent sodium channels but express voltage-dependent calcium channels, were also stably transfected with the c-fos-luc. GH4C1 cells expressing c-fos-luciferase were responsive to maitotoxin (1 ng ml−1) and a putative toxin produced by Pfiesteria piscicida. Although reporter gene assays are not designed to replace existing detection methods used to measure toxin activity in seafood, they do provide a valuable means to screen algal cultures for toxin activity, to conduct assay-guided fractionation and to characterize pharmacologic properties of algal toxins. Published in 1999 by John Wiley & Sons, Ltd.

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