Cyanobacteria are recognized producers of a wide array of toxic or otherwise bioactive secondary metabolites. The present study utilized the zebrafish ( Danio rerio) embryo as an aquatic animal model of vertebrate development to identify, purify and characterize lipophilic inhibitors of development ( i.e., developmental toxins) from an isolate of the freshwater cyanobacterial species, Aphanizomenon ovalisporum. Bioassay-guided fractionation led to the purification, and subsequent chemical characterization, of an apparent homologous series of isotactic polymethoxy-1-alkenes ( 1– 6), including three congeners ( 4– 6) previously identified from the strain, and two variants previously identified from other species ( 2 and 3), as well as one apparently novel member of the series ( 1). Five of the PMAs in the series ( 1– 5) were purified in sufficient quantity for comparative toxicological characterization, and toxicity in the zebrafish embryo model was found to generally correlate with relative chain length and/or methoxylation. Moreover, exposure of embryos to a combination of variants indicates an apparent synergistic interaction between the congeners. Although PMAs have been identified previously in cyanobacteria, this is the first report of their apparent toxicity. These results, along with the previously reported presence of the PMAs from several cyanobacterial species, suggest a possibly widespread distribution of the PMAs as toxic secondary metabolites and warrants further chemical and toxicological investigation.
References
[1]
Valério, E.; Chaves, S.; Tenreiro, R. Diversity and impact of prokaryotic toxins on aquatic environments: A review. Toxins 2010, 2, 2359–2410, doi:10.3390/toxins2102359.
[2]
Carmichael, W.W. A world overview-One-hundred-twenty-seven years of research on toxic cyanobacteria-Where do we go from here? Adv. Exp. Med. Biol. 2008, 619, 105–125, doi:10.1007/978-0-387-75865-7_4.
[3]
Ferr？o-Filho, A.; Kozlowsky-Suzuki, B. Cyanotoxins: Bioaccumulation and effects on aquatic animals. Mar. Drugs 2011, 9, 2729–2772, doi:10.3390/md9122729.
[4]
Cox, P.A.; Banack, S.A.; Murch, S.J.; Rasmussen, U.; Tien, G.; Bidigare, R.R.; Metcalf, J.S.; Morrison, L.F.; Codd, G.A.; Bergman, B. Diverse taxa of cyanobacteria produce β-methylamino-l-alanine, a neurotoxic amino acid. Proc. Natl. Acad. Sci. USA 2005, 102, 5074–5078.
[5]
Krüger, T.; M？nch, B.; Oppenh？user, S.; Luckas, B. LC-MS/MS determination of the isomeric neurotoxins BMAA (β-N-methylamino-l-alanine) and DAB (2,4-diaminobutyric acid) in cyanobacteria and seeds of Cycas revolute and Lathyrus latifolius. Toxicon 2010, 55, 547–557, doi:10.1016/j.toxicon.2009.10.009.
[6]
Berry, J.P.; Gantar, M.; Gawley, R.E.; Wang, M.; Rein, K.S. Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya Isolated from the Florida everglades. Comp. Biochem. Physiol. C Pharmacol. Toxicol. 2004, 139, 231–238.
[7]
Berry, J.; Gantar, M.; Gibbs, P.; Schmale, M. The zebrafish (Danio rerio) embryo as a model system for identification and characterization of developmental toxins from marine and freshwater microalgae. Comp. Biochem. Physiol. C Pharmacol. Toxicol. 2007, 145, 61–72, doi:10.1016/j.cbpc.2006.07.011.
[8]
Berry, J.; Gibbs, P.; Schmale, M.; Saker, M. Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo. Toxicon 2009, 53, 289–299, doi:10.1016/j.toxicon.2008.11.016.
[9]
Teraoka, H.; Dong, W.; Hiraga, T. Zebrafish as a novel experimental model for developmental toxicology. Congenit. Anom. 2003, 43, 123–132, doi:10.1111/j.1741-4520.2003.tb01036.x.
[10]
Hill, A.J.; Teraoka, H.; Heideman, W.; Peterson, R.E. Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol. Sci. 2005, 86, 6–19, doi:10.1093/toxsci/kfi110.
[11]
Wang, P.J.; Chien, M.S.; Wu, F.J.; Chou, H.N.; Lee, S.J. Inhibition of embryonic development by microcystin-LR in zebrafish, Danio rerio. Toxicon 2005, 45, 303–308, doi:10.1016/j.toxicon.2004.10.016.
[12]
Banker, R.; Carmeli, S.; Hadas, O.; Teltsch, B.; Porat, R.; Sukenik, A. Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophceae) isolated from Lake Kinneret, Israel. J. Phycol. 1997, 33, 613–616.
[13]
Banker, R.; Teltsch, B.; Sukenik, A.; Carmeli, S. 7-Epicylindrospermopsin, a toxic minor metabolite of the cyanobacterium Aphanizomenon ovalisporum from Lake Kinneret, Israel. J. Nat. Prod. 2000, 63, 387–389, doi:10.1021/np990498m.
[14]
Mynderse, J.S.; Moore, R.E. Isotactic polymethoxy-1-Alkenes from the blue-green alga Tolypothrix conglutinata var. chlorata. Phytochemistry 1979, 18, 1181–1183, doi:10.1016/0031-9422(79)80130-2.
[15]
Mori, Y.; Kohchi, Y.; Suzuki, M. Isotactic polymethoxy-1-alkenes from blue-green algae. Synthesis and absolute stereochemistry. J. Org. Chem. 1991, 56, 631–637, doi:10.1021/jo00002a027.
[16]
Mori, Y.; Kohchi, Y.; Noguchi, H.; Suzuki, M.; Carmeli, S.; Moore, R.E.; Patterson, G.M.L. Isotactic polymethoxy-1-alkenes from the terrestrial blue-green alga, Scytonema ocellatum: Structure and synthesis. Tetrahedron 1991, 47, 4889–4904, doi:10.1016/S0040-4020(01)80955-8.
Rao, M.R.; Faulkner, D.J. Isotactic polymethoxydienes from the Philippines sponge Myriastra clavosa. J. Nat. Prod. 2002, 65, 1201–1203, doi:10.1021/np020040b.
[19]
Le？o, P.; Pereira, A.R.; Liu, W.T.; Ng, J.; Pevzner, P.A.; Dorrestein, P.C.; K？nig, G.M.; Vasconcelos, V.M.; Gerwick, W.H. Synergistic allelochemicals from a freshwater cyanobacterium. Proc. Natl. Acad. Sci. USA 2010, 107, 11183–11188.
[20]
MacMillan, J.B.; Ernst-Russell, M.A.; de Ropp, J.S.; Molinski, T.F. Lobocyclamides A-C, lipopeptides from a cryptic cyanobacterial mat containing Lyngbya confervoides. J. Org. Chem. 2002, 67, 8210–8215, doi:10.1021/jo0261909.
[21]
Gantar, M.; Berry, J.P.; Thomas, S.; Wang, M.; Perez, R.; Rein, K.S. Allelopathic activity among cyanobacteria and microalgae isolated from Florida freshwater habitats. FEMS Microbiol. Ecol. 2008, 64, 55–64, doi:10.1111/j.1574-6941.2008.00439.x.
[22]
Brand, M.; Granato, M.; Nüsslein-Volhard, C. Keeping and Raising Zebrafish. In Zebrafish; Nüsslein-Volhard, C., Dahm, R., Eds.; Oxford University Press: Oxford, UK, 2002; pp. 7–37.
