Brevetoxins (PbTx) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis, an organism linked to periodic red tide blooms. 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 through the same routes of exposure. Evidence suggests that respiratory exposure to brevetoxins can lead to a severe inflammatory response. 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. The goal of the current research project is to determine how these marine toxins evoke an immune response, specifically by examining the effects of brevetoxin and brevetoxin antagonists on T cells. The hypothesis is that brevetoxins alter cell proliferation, cause DNA damage, disrupt normal signal transduction, and ultimately cause cell death, possibly through an apoptotic mechanism, and the natural and synthetic brevetoxin antagonists are able to prevent and reverse these deleterious effects, making them a useful pharmaceutical agent not only for brevetoxin exposure treatment, but also for human diseases exhibiting similar inflammatory congestion, like Asthma and Cystic Fibrosis. The effects of brevetoxins and antagonists were determined by cell proliferation assays, comet assays and examination of caspase activation, Poly ADP- ribose polymerase (PARP) cleavage and gene expression. Exposure of Jurkat E6-1 Cells to Brevetoxin 2, 3, 6, and 9 at doses of 10-5M severely inhibited proliferation. Further analysis revealed positive staining for apoptosis, PARP cleavage, caspase 3/7 and caspase 8 activation, decreased intracellular sodium and increased intracellular calcium concentrations. PCR array gene expression analysis revealed significant change in gene expression. The apoptosis PCR array yielded 30 up-regulated and 4 down-regulated genes divided into the following families: TNF ligand, TNF receptor, Bcl-2, caspase, IAP, CARD, death domain, CIDE, p53 and DNA damage response, and anti-apoptosis. The DNA Damage Signaling Pathway Array had 20 up-regulated genes and 5 down-regulated genes from the following families: apoptosis, cell cycle arrest, cell cycle checkpoint, DNA repair-damaged DNA binding, DNA repair-double strand break repair, DNA repair-mismatch repair, and other genes related to DNA repair. The Common Cytokine PCR array had 50 up-regulated genes from the following gene families: interferons, interleukins, bone morphogenic protein and TGF-ï¢, PDGF/VEGF, TNF Superfamily, and other growth factors/cytokines. Exposure of Jurkat E6-1 cells to brevetoxin in combination with the antagonists brevenal, alpha naphthoyl, and beta naphthoyl did not improve cell proliferation, which is in stark contrast to experiments conducted with CHO-K1 BH4 cells. This disparity may be due to the difference in voltage gated sodium channel subtypes that these cells possess. The results demonstrate that brevetoxins can induce apoptosis and DNA damage and that the antagonists may show selectivity based on VGSC subtype.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-07302008-152733 |
| Date | 09 December 2008 |
| Creators | Murrell, Rachel Nichole |
| Contributors | Dr. James E. Gibson, Dr. Patricia McClellan-Green, Dr. Damian Shea, Dr. Scott Laster |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-07302008-152733/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0019 seconds