Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, and ciguatoxins.
In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.
Manger
R L
RL
Seafood Products Research Center, United States Food and Drug Administration, Bothell, Washington 98041-3012.
Leja
L S
LS
Lee
S Y
SY
Hungerford
J M
JM
Wekell
M M
MM
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002922:Ciguatoxins / Q000032:analysis* / Q000633:toxicity; D008387:Marine Toxins / Q000032:analysis* / Q000633:toxicity; D051379:Mice; D009447:Neuroblastoma; D009498:Neurotoxins / Q000032:analysis* / Q000633:toxicity; D035964:Oxocins; D012530:Saxitoxin / Q000032:analysis* / Q000633:toxicity; D015222:Sodium Channels / Q000187:drug effects* / Q000502:physiology; D013778:Tetrazolium Salts; D014407:Tumor Cells, Cultured; D000095702:Polyether Toxins
D002922:Ciguatoxins; D008387:Marine Toxins; D009498:Neurotoxins; D035964:Oxocins; D012530:Saxitoxin; D015222:Sodium Channels; D013778:Tetrazolium Salts; C053342:brevetoxin; D000095702:Polyether Toxins