title: Brevetoxins 2, 3, 6, and 9 show variability in potency and cause significant induction of DNA damage and apoptosis in Jurkat E6-1 cells.
abstract: Brevetoxins (PbTx) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis, an organism linked to periodic red tide blooms. Brevetoxins exert their toxicity by interacting with neurotoxin receptor site five associated with domain IV of the alpha subunit of the voltage gated sodium channel. Brevetoxin binding to tissues that contain voltage gated sodium channels on excitable cells results in membrane depolarization, repetitive firing, and increase in sodium currents. Brevetoxins have been linked to deaths in marine mammals, which are exposed through ingestion of organisms harboring high brevetoxin concentrations and through the inhalation of aerosolized brevetoxins. Humans are also at risk, primarily through respiratory exposure which can result in a severe inflammatory response. The purpose of this study was to determine the effect of four brevetoxins on Jurkat E6-1 cell proliferation, to assess their variability in potency, genotoxicity, and to determine if brevetoxin causes cell death, specifically through an apoptotic or necrotic mechanism. PbTx 2, 3, 6, and 9 were tested at concentrations of 10(-4)-10(-12) M to determine the IC(50) values and effect on cell proliferation. The IC(50) concentration was then used in the single cell gel electrophoresis assay to determine genotoxicity. The ability to induce apoptosis was then assessed with the Vybrant apoptosis assay, caspase activation assays and PARP cleavage. Results from the cellular proliferation assays demonstrated that high doses of PbTxs inhibit the ability of Jurkat cells to proliferate while lower doses caused an increase in proliferation and that PbTx2 is the most cytotoxic brevetoxin followed by brevetoxins 6, 3, and 9. Brevetoxins 2, 3, and 6 all caused significant DNA damage. A 4 h exposure to brevetoxins 2, 3, 6, and 9 at values close to the IC(50) values resulted in apoptosis positive staining in Jurkat E6-1 cells. High doses of brevetoxins 2 and 6 resulted in activation of caspases 3/7 and 8 and cleavage of poly (ADP-ribose) polymerase (PARP). The conclusions are that brevetoxins affect cell proliferation in a dose-dependent fashion, are genotoxic, and cause cell death through an apoptotic mechanism.
authors.affiliation: Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, 27695-7633, USA. murrellr@ecu.edu
authors.forename: Rachel N
authors.initials: RN
authors.lastname: Murrell
authors.forename: James E
authors.initials: JE
authors.lastname: Gibson
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D019169:Jurkat Cells; D008387:Marine Toxins / Q000008:administration & dosage / Q000633:toxicity*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000008:administration & dosage / Q000633:toxicity; D035964:Oxocins / Q000008:administration & dosage / Q000633:toxicity*; D013997:Time Factors; D000095702:Polyether Toxins
chemical_list: D020169:Caspases; D008387:Marine Toxins; D009153:Mutagens; D035964:Oxocins; C053342:brevetoxin; D000095702:Polyether Toxins