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 IC50 values and effect on cell proliferation. The IC50 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 IC50 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.
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References
Abraham WM, Bourdelais AG, Baden DG (2002) Polyether brevetoxin (PbTx) aerosols cause bronchoconstriction in sheep. Am J Respir Crit Care Med 165:A20
Abraham WM, Bourdelais AJ, Sabater JR, Ahmed A, Lee TA, Serebriakov I, Baden DG (2005) Airway response to aerosolized brevetoxins in an animal model of asthma. Am J Respir Crit Care Med 171:26–34. doi:10.1164/rccm.200406-735OC
Ashkenazi A (2008) Targeting the extrinsic apoptosis pathway in cancer. Cytokine Growth F R 19:325–331. doi:10.1016/j.cytogfr.2008.04.001
Azad N, LaPaglia N, Kirsteins L, Uddin S, Steiner J, Williams DW, Lawrence AM, Emanuele NV (1997) Jurkat cell proliferative activity is increased by luteinizing hormone-releasing hormone. J Endocrinol 153:241–249. doi:10.1677/joe.0.1530241
Boatright KM, Salvesen GS (2003) Mechanisms of caspase activation. Curr Opin Cell Biol 15:725–731. doi:10.1016/j.ceb.2003.10.009
Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P, Blenis J, Tschopp J (2000) TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2:241–243. doi:10.1038/35008667
Bossart GD, Baden DG, Ewing RY, Roberts B, Wright SD (1998) Brevetoxicosis in Manatees (Trichechus Manatus latirostris) from the 1996 Epizootic: gross, histologic, and immunohistochemical features. Toxicol Pathol 26:276–282
Bourdelais AJ, Campbell S, Jacocks H, Naar J, Wright JL, Carsi J, Baden DG (2004) Brevenal is a natural inhibitor of Brevetoxin action in sodium channel receptor binding assays. Cell Mol Neurobiol 24:553–563. doi:10.1023/B:CEMN.0000023629.81595.09
Bourdelais AJ, Jacocks HM, Wright JL, Bigwarfe PM Jr, Baden DG (2005) A new polyether ladder compound produced by the dinoflagellate Karenia brevis. J Nat Prod 68:2–6. doi:10.1021/np049797o
Casiano CA, Ochs RL, Tan EM (1998) Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes. Cell Death Differ 5:183–190. doi:10.1038/sj.cdd.4400336
Catterall WA (1992) Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev 72(4 Supplement):S15–S48
Czene S, Testa E, Nygren J, Belyaev I, Harms-Ringdahl M (2002) DNA fragmentation and morphological changes in apoptotic human lymphocytes. Biochem Biophys Res Commun 294:872–878. doi:10.1016/S0006-291X(02)00588-0
Fairclough L, Urbanowicz RA, Corne J, Lamb JR (2008) Killer cells in chronic obstructive pulmonary disease. Clin Sci 114:533–541. doi:10.1042/CS20070356
Fleming LE, Kirkpatrick B, Backer LC, Bean JA, Wanner A, Dalpra D, Tamer R, Zaias J, Cheng YS, Pierce R, Naar J, Abraham W, Clark R, Zhou Y, Henry MS, Johnson D, Van De Bogart G, Bossart GD, Harrington M, Baden DG (2005) Initial evaluation of the effects of aerosolized Florida red tide toxins (brevetoxins) in persons with asthma. Environ Health Perspect 113:650–657
Gawley RE, Rein KS, Jeglitsch G, Adams DJ, Theodorakis EA, Tiebes J, Nikolaou KC, Baden DG (1995) The relationship of brevetoxin ‘length’ and A-ring functionality to binding and activity in neuronal sodium channels. Chem Biol 2:533–541. doi:10.1016/1074-5521(95)90187-6
Hubeau C, Le Naour R, Abely M, Hinnrasky J, Guenounou M, Gaillard D, Puchelle E (2004) Dysregulation of IL-2 and IL-8 production in circulating T lymphocytes from young cystic fibrosis patients. Clin Exp Immunol 135:528–534. doi:10.1111/j.1365-2249.2003.02385.x
Jeglitsch G, Rein K, Baden DG, Adams DG (1998) Brevetoxin-3 (PbTx-3) and its derivatives modulate single tetrodotoxin-sensitive sodium channels in rat sensory neurons. J Pharmacol Exp Ther 284:516–525
Jin Z, El-Deiry WS (2005) Overview of cell death signaling pathways. Cancer Biol Ther 4:139–163
Juo P, Kuo CJ, Yuan J, Blenis J (1998) Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr Biol 8:1001–1008. doi:10.1016/S0960-9822(07)00420-4
Kirkpatrick B, Fleming LE, Squicciarini D, Backer LC, Clark R, Abraham W, Benson J, Cheng YS, Johnson D, Pierce R, Zaias J, Bossart GD, Baden DG (2004) Literature review of Florida red tide: implications for human health effects. Harmful Algae 3:99–115. doi:10.1016/j.hal.2003.08.005
Krammer PH, Arnold R, Lavrik IN (2007) Life and death in peripheral T cells. Nat Rev Immunol 7:532–542. doi:10.1038/nri2115
Lai Z, Chen Y, Nishimura Y, Nishi K (2000) An amiloride-sensitive and voltage-dependent Na+ channel in an HLA-DR-restricted human T cell clone. J Immunol 165:83–90
Lakhani SA, Masud A, Kuida K, Porter GA Jr, Booth CJ, Mehal WZ, Inayat I, Flavell RA (2006) Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311:847–851. doi:10.1126/science.1115035
Lankoff A, Carmichael WW, Grasman KA, Yuan M (2004) The uptake kinetics and immunotoxic effects of microcystin-LR in human and chicken peripheral blood lymphocytes in vitro. Toxicology 204:23–40. doi:10.1016/j.tox.2004.05.016
Le Hegarat L, Jacquin A, Bazin E, Fessard V (2005) Genotoxicity of the Marine Toxin Okadaic Acid, in Human Caco-2 Cells and in Mice Gut Cells. Environ Toxicol 21:55–64. doi:10.1002/tox.20154
Le Rhun Y, Kirkland JB, Shah GM (1998) Cellular responses to DNA damage in the absence of Poly (ADP-ribose) polymerase. Biochem Biophys Res Commun 245:1–10. doi:10.1006/bbrc.1998.8257
Lee E, Oh E, Lee J, Sul D, Lee J (2004) Use of the tail moment of lymphocytes to evaluate DNA damage in human biomonitoring studies. Toxicol Sci 81:121–132. doi:10.1093/toxsci/kfh184
Lewis RS (2003) Calcium oscillations in T-cells: mechanisms and consequences for gene expression. Biochem Soc Trans 31:925–929. doi:10.1042/BST0310925
Malanga M, Althaus FR (2005) The role of poly (ADP-ribose) in the DNA damage signaling network. Biochem Cell Biol 83:354–364. doi:10.1139/o05-038
Mattson MP (2008) Hormesis defined. Ageing Res Rev 7:1–7. doi:10.1016/j.arr.2007.08.007
Medoff BD, Thomas SY, Luster AD (2008) T cell trafficking in allergic asthma: the ins and outs. Annu Rev Immunol 26:205–232. doi:10.1146/annurev.immunol.26.021607.090312
Milian A, Nierenberg K, Fleming LE, Bean JA, Wanner A, Reich A, Backer LC, Jayroe D, Kirkpatrick B (2007) Reported respiratory symptom intensity in asthmatics during exposure to aerosolized Florida red tide toxins. J Asthma 44:583–587. doi:10.1080/02770900701539251
Morita T, Nagaki T, Fukuda I, Okumura K (1992) Clastogenicity of low pH to various cultured mammalian cells. Mutat Res 272:223–226
Nelms BE, Moravec R, Riss T (1997) Measuring apoptosis in individual cells with the comet assay. Promega Notes 64:13–17
Peter ME, Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 10:26–35. doi:10.1038/sj.cdd.4401186
Roselli F, Livrea P, Jirillo E (2006) Voltage-gated sodium channel blockers as immunomodulators. Recent Pat CNS Drug Discov 1:83–91. doi:10.2174/157488906775245255
Sayer A, Hu Q, Bourdelais AJ, Baden DG, Gibson JE (2005) The effect of brevenal on brevetoxin-induced DNA damage in human lymphocytes. Arch Toxicol 79:683–688. doi:10.1007/s00204-005-0676-2
Sayer A, Hu Q, Bourdelais AJ, Baden DG, Gibson JE (2006) The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro. Food Chem Toxicol 44:1082–1091. doi:10.1016/j.fct.2006.01.002
Schreibmayer W, Jeglitsch G (1992) The sodium channel activator Brevetoxin-3 uncovers a multiplicity of different open states of the cardiac sodium channel. Biochim Biophys Acta 1104:233–242. doi:10.1016/0005-2736(92)90035-K
Shen Y, White E (2001) p53 dependent apoptosis pathways. Adv Cancer Res 82:55–84. doi:10.1016/S0065-230X(01)82002-9
Sprick MR, Weigand MA, Reiser E, Rauch CT, Juo P, Blenis J, Krammer PH, Walczak H (2000) FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 12:599–609. doi:10.1016/S1074-7613(00)80211-3
Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281:1312–1316. doi:10.1126/science.281.5381.1312
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221. doi:10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
Trainer VL, Baden DG, Catterall WA (1994) Identification of peptide components of the brevetoxin receptor site of rat brain sodium channels. J Biol Chem 269:19904–19909
Varfolomeev EE, Schuchmann M, Luria V, Chiannilkuchai N, Beckmann JS, Mett IL, Rebtikov D, Brodianski VM, Kemper OC, Kollet O, Lapidot T, Soffer D, Sobe T, Avraham KB, Gonchatov T, Holtmann H, Lonai P, Wallach D (1998) Targeted disruption of the mouse caspase 8 gene ablates cell death induction by the TNF receptors, FAS/apo1, and dr3 and is lethal prenatally. Immunity 9:267–276. doi:10.1016/S1074-7613(00)80609-3
Walsh CJ, Luer CA, Noyes DR (2005) Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris. Vet Immunol Immunopathol 103:247–256. doi:10.1016/j.vetimm.2004.09.026
Walsh CJ, Leggett SR, Strohbehn K, Pierce RH, Sleasman JW (2008) Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat). Mar Drugs 6:291–307
Acknowledgments
This research was supported by Marine Biotechnology in North Carolina and the North Carolina Agromedicine Center and USDA/CSREES. The PbTxs were provided under NIEHS grant PO1 ES10594. The authors would like to extend their thanks to Daniel Baden for providing brevetoxins, to Jackie Masterson for cell culture assistance and Rukiyah Van Dross, Pranita Katwa, and Christian Kuc for their assistance with the PARP experiment.
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Murrell, R.N., Gibson, J.E. Brevetoxins 2, 3, 6, and 9 show variability in potency and cause significant induction of DNA damage and apoptosis in Jurkat E6-1 cells. Arch Toxicol 83, 1009–1019 (2009). https://doi.org/10.1007/s00204-009-0443-x
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DOI: https://doi.org/10.1007/s00204-009-0443-x