The isolation and structure elucidation of two cyclic peptides, pahayokolides A (1) and B (2), is described. Structural features determined for these compounds include a pendent N-acetyl-N-methyl leucine, both E- and Z-dehydrobutyrines, a homophenylalanine, and an unusual polyhydroxy amino acid that is most likely of mixed polyketide synthase/nonribosomal peptide synthase origin. These peptides were purified from a new species of cyanobacteria of the genus Lyngbya, which was isolated from a periphyton mat from the Florida Everglades.

The hepatotoxin okadaic acid (OA) was incubated with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Both CYP3A4 and CYP3A5 converted OA to a mixture of the same four metabolites, but incubation with CYP3A4 resulted in higher levels of conversion. Michaelis-Menten parameters, K m (73.4 μM) and V max (7.23 nmol of metabolites nmol -1 min -1 ) for CYP3A4 were calculated by analyzing double-reciprocal plots. LC-MS n analysis and chemical interconversion indicate that metabolites 2 and 3 are the 11S-hydroxy and 11R-hydroxy okadaic acid respectively, while metabolite 4 is 11-oxo okadaic acid. LC-MS n analysis of metabolite 1 shows a molecular ion which corresponds to an addition of 16 amu to OA, also suggesting hydroxylation, but the specific site has not been identified. The same four metabolites were produced upon incubation of okadaic acid with pooled human liver microsomes. This transformation could be completely inhibited with ketokonazole, and inhibitor of the CYP3A family of enzymes. The metabolites were determined to be only slightly less potent inhibitors of serine threonine protein phosphatase 2A (PP2A) when compared to OA. As PP2A is the principle molecular target for OA, these oxidative transformations may not effectively detoxify OA.

Extracts of fifty-seven newly isolated strains of dinoflagellates and raphidophytes were screened for protein phosphatase (PP2A) inhibition. Five strains, identified by rDNA sequence analysis as Prorocentrum rhathymum, tested positive and the presence of okadaic acid was confirmed in one strain by HPLC-MS/MS and by HPLC with fluorescence detection and HPLC-MS of the okadaic acid ADAM derivative. Quantitation of the ADAM derivative indicated that the concentration of okadaic acid in the culture medium is 0.153 μg/L. KeywordsProrocentrum rhathymum; okadaic acid; diarrheic shellfish poisoning (DSP) toxins; LC-MS; ADAM derivative; HPLC-FLD The suite of marine toxins that includes okadaic (OA) acid and the dinophysistoxins (DTX) is collectively known as DSP (diarrheic shellfish poisoning) toxins. The acute symptoms of DSP include diarrhea, nausea, vomiting and abdominal pain. Outbreaks have been documented worldwide and are associated with the consumption of mussels, scallops, or clams tainted with OA, its analogs or derivatives. (Gestal-Otero, 2000). OA has been isolated from several dinoflagellates in the genera Prorocentrum and Dinophysis, including the species P. lima (Yasumoto et al., 1987), P. hoffmanianum (Murakami et al., 1982), P. concavum (Dickey et al., 1990), P. maculosum (Zhou and Fritz, 1993), P. belizeanum , P. faustiae (Morton 1998), P. arenarium (Ten-Hage et al., 2000), D. acuta (Draisci et al., 1998) and D. fortii (Murata et al., 1982). In addition to OA, several related polyethers were isolated from these dinoflagellates, including the dinophysis toxins, DTX-1 and DTX-2 which differ in the location and number of methyl substituents (Murata et al., 1982;Yasumoto, et al., 1985;Hu et al., 1993). OA, DTX-1 and DTX-2 are inhibitors of protein phosphatases PP1 and PP2A (Dounay and Forsyth, 2002 We recently reported the isolation of over fifty strains of dinoflagellates by viable high speed single-cell sorting ). Five of these strains (6-9 and 25) matched most closely with P. rhathymum in a BLAST (Altschul et al., 1990) analysis of their large-subunit ribosomal genes ) and tested positive for protein phosphatase (PP2A) inhibition in preliminary screening. The presence of OA was confirmed in one strain by HPLC with fluorescent detection of the ADAM (9-anthryldiazomethane) derivative (Quilliam et al., 1998) and by HPLC-MS and MS/MS experiments.Fifty-seven field strains were sequenced in the D1/D2 region of the LSU rDNA and compared with GenBank sequence data ). Dinoflagellate and raphidophyte genera identified included Akashiwo, Amphidinium, Heterocapsa, Cachonina, Chatonella, Coolia, Fragilidinium, Heterosigma, Karlodinium, Karenia, Protoceratium, Scripsciella and Prorocentrum. Field strains 6, 7, 8, 9, 23, 25 and identified strains CCMP687 (Prorocentrum mexicanum) and CCMP1591 (Prorocentrum micans) clustered within a clade of the genus Prorocentrum (Fig. 1). The sequences for strains 6, 7, 8 and 9 were identical to each other and matched with two sequences (AY259166 and AY259167) of P. rhathymu...

Four metabolites were identified upon incubation of brevetoxin (PbTx-2) with human liver microsomes. Chemical transformation of PbTx-2 confirmed the structures of three known metabolites BTX-B5, PbTx-9 and 41, 43-dihydro-BTX-B5 and a previously unknown metabolite, 41, 43-dihydro-PbTx-2. These metabolites were also observed upon incubation of PbTx-2 with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Cytochrome P450 3A4 produced oxidized metabolites while other CYPs generated the reduced products. Keywordsbrevetoxins; PbTx-2; metabolism; cytochrome P450; human liver microsome Brevetoxins, produced by the "Florida red tide" dinoflagellate Karenia brevis, are a suite of neurotoxins which are responsible for massive fish kills and marine mammal mortalities (Landsberg et al., 2009). In humans, the brevetoxins are responsible for a syndrome known as Neurotoxic Shellfish Poisoning (NSP) which results from the consumption of tainted shellfish (Watkins et al., 2008) as well as respiratory distress in beachgoers upon exposure to aerosolized toxins (Fleming et al., 2005). Brevetoxin PbTx-2 (Fig. 1) is the primary constituent of this suite of toxins.Several studies of brevetoxin excretion and metabolism in mammals have demonstrated that the hepatobiliary system plays a key role in the detoxification and elimination of brevetoxins. Radwan found that in rats, PbTx-2 is rapidly detoxified and excreted in urine as cysteine conjugates and an unidentified oxidized metabolite. (Radwan et al., 2005) On the other hand, when fed to rats, [ 3 H]-PbTx-3, which is unable to form cysteine conjugates, is eliminated in both the urine and feces. (Poli, et al., 1990;Cattet and Geraci, 1993 Conflict of interestThe authors declare that there are no conflicts of interest.Supplementary information: Supplementary data associated with this article including experimental details may be found at XXXXXXX Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptToxicon. Author manuscript; available in PMC 2011 September 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript brevetoxin-like activity which was later confirmed by LC-MS to be due to the presence of PbTx-3 and other unidentified metabolites. (Poli et al., 2000) Wang reported the conversion of PbTx-2 to PbTx-3 and the hydrolyzed A-ring lactone by rat liver microsomes. Later Radwan examined the metabolic activities of purified cDNA-expressed rat liver cytochrome P-450 (CYP) enzymes toward PbTx-2. All six CYPs studied (CYP1A2, CYP2A2, CYP2C11, CYP2D1...