Document Detail


A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line.
MedLine Citation:
PMID:  18023406     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.
Authors:
Mirella Bellocci; Giuseppe Ronzitti; Anna Milandri; Nunzia Melchiorre; Claudio Grillo; Roberto Poletti; Takeshi Yasumoto; Gian Paolo Rossini
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-11-20
Journal Detail:
Title:  Analytical biochemistry     Volume:  374     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2008 Mar 
Date Detail:
Created Date:  2008-02-01     Completed Date:  2008-04-10     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  48-55     Citation Subset:  IM    
Affiliation:
Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, I-41100 Modena, Italy.
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MeSH Terms
Descriptor/Qualifier:
Acrylamides / analysis*,  pharmacology
Animals
Bicyclo Compounds, Heterocyclic / analysis,  pharmacology
Cell Line, Tumor
Cell Survival
Cytotoxicity, Immunologic*
Humans
Mytilus
Ouabain / pharmacology
Pyrans / analysis,  pharmacology
Sea Urchins
Chemical
Reg. No./Substance:
0/Acrylamides; 0/Bicyclo Compounds, Heterocyclic; 0/Pyrans; 0/ostreocin D; 11077-03-5/palytoxin; 630-60-4/Ouabain

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