Reporter gene assays for algal-derived toxins.
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conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method.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.
Fairey
E R
ER
Marine Biotoxins Program, NOAA-National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412, USA.
Ramsdell
J S
JS
D000818:Animals; D001681:Biological Assay; D002922:Ciguatoxins / Q000032:analysis / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000187:drug effects* / Q000235:genetics; D016762:Genes, fos; D008156:Luciferases / Q000235:genetics; D008387:Marine Toxins / Q000032:analysis / Q000633:toxicity*; D051379:Mice; D009447:Neuroblastoma; D009498:Neurotoxins / Q000032:analysis / Q000633:toxicity*; D035964:Oxocins; D020045:Pfiesteria piscicida / Q000235:genetics / Q000378:metabolism; D010902:Pituitary Gland / Q000187:drug effects / Q000473:pathology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012530:Saxitoxin / Q000032:analysis / Q000633:toxicity; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology; D000095702:Polyether Toxins
D002922:Ciguatoxins; D008156:Luciferases; D008387:Marine Toxins; D009498:Neurotoxins; D035964:Oxocins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D012530:Saxitoxin; C053342:brevetoxin; D000095702:Polyether Toxins; C020394:maitotoxin