title: Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin.
abstract: The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.
authors.affiliation: Department of Clinical and Experimental Pharmacology, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: S M
authors.initials: SM
authors.lastname: Froscio
authors.forename: A R
authors.initials: AR
authors.lastname: Humpage
authors.forename: P C
authors.initials: PC
authors.lastname: Burcham
authors.forename: I R
authors.initials: IR
authors.lastname: Falconer
mesh_terms: D000470:Alkaloids; D000818:Animals; D001427:Bacterial Toxins; D001681:Biological Assay / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000737:chemistry*; D000087522:Cyanobacteria Toxins; D004305:Dose-Response Relationship, Drug; D013058:Mass Spectrometry; D011500:Protein Synthesis Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D012680:Sensitivity and Specificity; D014498:Uracil / Q000031:analogs & derivatives* / Q000032:analysis* / Q000494:pharmacology*
chemical_list: D000470:Alkaloids; D001427:Bacterial Toxins; D000087522:Cyanobacteria Toxins; D011500:Protein Synthesis Inhibitors; C089595:cylindrospermopsin; D014498:Uracil