title: A rapid and sensitive assay for paralytic shellfish poison (PSP) toxins using mouse brain synaptoneurosomes.
abstract: A membrane potential assay using mouse brain synaptoneurosomes was evaluated for the determination of paralytic shellfish poison (PSP) toxin content of mussels and other bivalve species important to the shellfish industry. The assay relies on the ability of PSP toxins to block veratridine-induced depolarization of synaptoneurosomes. Changes in the membrane potential of synaptoneurosomes were monitored using the voltage-sensitive fluorescent probe rhodamine 6G. Standard saxitoxin was found to be a potent inhibitor of the membrane depolarizing effects of the sodium channel activator veratridine (I(50) ca. 4 nM). Likewise, shellfish extracts containing PSP toxins inhibited veratridine-induced depolarization. Neither saxitoxin or shellfish extracts had any discernible effect on the resting membrane potential of synaptoneurosomes. When synaptoneurosomal results for extracts of mussels (n=120) and other shellfish (n=29) were correlated with official mouse toxicity assay data there was very good agreement (r(2)=0.84 and 0.86, respectively), indicating that the in vitro assay has utility for a variety of commercially relevant shellfish species. Our investigation suggests that the mouse synaptoneurosome assay is of similar sensitivity to the official CD1 mouse toxicity assay. The synaptoneurosome fraction can be prepared quickly (approx. 40 min) and an individual assay takes less than 7 min. Since 20 such assays can be performed using material from a single CD1 mouse brain, there is considerable opportunity for reducing the number of animals required in conventional PSP monitoring while retaining the same animal system.
authors.affiliation: Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6. nicholso@sfu.ca
authors.forename: Russell A
authors.initials: RA
authors.lastname: Nicholson
authors.forename: Guo Hua
authors.initials: GH
authors.lastname: Li
authors.forename: Enrico
authors.initials: E
authors.lastname: Buenaventura
authors.forename: David
authors.initials: D
authors.lastname: Graham
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D008974:Mollusca / Q000737:chemistry*; D015203:Reproducibility of Results; D012235:Rhodamines; D012530:Saxitoxin / Q000032:analysis* / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000032:analysis*; D013574:Synaptosomes / Q000187:drug effects* / Q000502:physiology; D014701:Veratridine / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; C026188:rhodamine 6G; D012530:Saxitoxin; D014701:Veratridine