Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.
A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.
Leira
F
F
ANFACO-CECOPESCA, Campus Universitario de Vigo, Vigo 36310, Spain. fleira@anfaco.cesga.es
Alvarez
C
C
Cabado
A G
AG
Vieites
J M
JM
Vieytes
M R
MR
Botana
L M
LM
D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D049872:Bivalvia / Q000737:chemistry; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000032:analysis / Q000494:pharmacology; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D008387:Marine Toxins / Q000032:analysis* / Q000494:pharmacology; D000432:Methanol / Q000494:pharmacology; D051379:Mice; D008978:Mollusk Venoms; D019319:Okadaic Acid / Q000494:pharmacology; D035964:Oxocins / Q000032:analysis / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results; D012530:Saxitoxin / Q000032:analysis / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012758:Shellfish
D000199:Actins; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D008387:Marine Toxins; D008978:Mollusk Venoms; D035964:Oxocins; D019319:Okadaic Acid; D012530:Saxitoxin; D010749:Phosphoprotein Phosphatases; C012301:domoic acid; C066632:yessotoxin; D007608:Kainic Acid; D000432:Methanol