pectenotoxin-2 and pectenotoxin-1

An enzyme capable of hydrolysing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the Greenshell™ mussel Perna canaliculus was isolated and characterized. The enzyme was purified by sequential polyethylene glycol fractionation, anion exchange, hydrophobic interaction, gel filtration and hydroxyapatite chromatography. The enzyme was an acidic (pI ∼ 4.8), monomeric, 67 kDa, serine esterase with optimum activity at pH 8.0 and 25 °C. PTX2 and PTX1 were hydrolysed but the enzyme was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolysed short and medium chain length (C2-C10) 4-nitrophenyl-esters, okadaic acid C8-C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). MALDI-Tof MS/MS analysis showed that the enzyme had some homology with a juvenile hormone esterase from the Red Flour Beetle Tribolium castaneum, although BLAST searches of several data bases using de novo amino acid sequences failed to identify any homology with known proteins.

Topics: Amino Acid Sequence; Animals; Carboxylic Ester Hydrolases; Esterases; Furans; Hepatopancreas; Hydrogen-Ion Concentration; Insect Proteins; Kinetics; Macrolides; Marine Toxins; Molecular Weight; New Zealand; Okadaic Acid; Peptide Fragments; Perna; Pyrans; Sequence Homology; Species Specificity; Substrate Specificity; Tribolium

Conditions for the determination of lipophilic marine toxins, such as yessotoxins and pectenotoxins (PTX)-6, were investigated with capillary electrophoresis coupled to mass spectrometry (MS) with an electrospray ionization source. After optimization, a simple and MS compatible alkaline volatile buffer solution of ammonium acetate was selected as background electrolyte, with isopropanol/water (80/20, v/v) sheath liquid modified with ammonium acetate used at the electrospray ionization (ESI) source. Previously to capillary electrophoresis (CE) separations, the application of an on-line sample pre-concentration approach based on field-amplified sample stacking was accomplished to increase sensitivity. As a result, the limits of detection provided by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) were 0.02 microg ml(-1) (0.01 microg g(-1)), which corresponded to 1.25 pg for yessotoxin and 0.25 microg ml(-1) (0.13 microg g(-1) and 13.25 pg on capillary) for PTX-6. Accuracy tests showed 97.7% recovery from spiked blank mussel samples that showed no significant matrix influence running under optimal conditions. Intermediate precision was close to 4% relative standard deviation (RSD) for the migration time, and an RSD of 7.5% for peak areas. The method was successfully applied to naturally contaminated seafood samples in which yessotoxins and pectenotoxins-6 were clearly determined. This work demonstrated the potential of CE-ESI-MS to be applied for a sensitive determination of lipophilic toxins from the marine environment as alternative to liquid chromatography-electrospray ionization-single quadrupole mass spectrometry (LC-ESI-MS) for this purpose.

Topics: Animals; Furans; Macrolides; Marine Toxins; Mollusk Venoms; Oxocins; Pyrans; Sensitivity and Specificity; Shellfish; Spain; Spectrometry, Mass, Electrospray Ionization

Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity.. Effects of pectenotoxin (PTX)-1, PTX-2, PTX-2 seco acid (PTX-2SA) and PTX-11 were measured in a hepatocyte cell line with cancer cell characteristics (Clone 9) and in primary cultures of rat hepatocytes. Cell morphology was assessed by confocal microscopy; F- and G-actin were selectively stained and cell viability measured by Alamar Blue fluorescence.. Clone 9 cells and primary hepatocytes showed a marked depolymerization of F-actin with PTX-1, PTX-2 and PTX-11 (1-1000 nM) associated with an increase in G-actin level. However, morphology was only clearly altered in Clone 9 cells. PTX-2SA had no effect on the actin cytoskeleton. Despite the potent F-actin depolymerizing effect, PTX-1, PTX-2 or PTX-11 did not decrease the viability of Clone 9 cells after 24-h treatment. Only prolonged incubation (> 48 h) with PTXs induced a fall in viability, and under these conditions, morphology of both Clone 9 and primary hepatocytes was drastically changed.. Although the actin cytoskeleton was clearly altered by PTX-1, PTX-2 and PTX-11 in the hepatocyte cell line and primary hepatocytes, morphological assessments indicated a higher sensitivity of the cancer-like cell line to these toxins. However, viability of both cell types was not altered.

Topics: Actins; Animals; Cells, Cultured; Clone Cells; Cytoskeleton; Fluorescent Dyes; Furans; Hepatocytes; Macrolides; Male; Microscopy, Confocal; Phalloidine; Pyrans; Rats; Rats, Sprague-Dawley; Xanthenes

Pectenotoxins are a group of natural products from marine origin that can accumulate in shellfish and intoxicate humans. Recently, novel homologues such as pectenotoxin-11 (PTX-11) and pectenotoxin-2 seco acid (PTX-2SA) have been identified. Their toxic potential towards experimental animals has been evaluated however their interaction with cellular systems is almost unknown. This is the first report showing (i) the biological activity of PTX-11 and PTX-2SA on actin cytoskeleton and morphology of living cells and (ii) the structure- activity relationship for this family of toxic compounds.. Fluorescent phalloidin was utilized to quantify and visualize any modification in polymerized actin. Fluorescence values were obtained with laser-scanning cytometer and cells were imaged through confocal microscopy. For structure-activity evaluations, pectenotoxin-1 (PTX-1) and pectenotoxin-2 (PTX-2) was also analyzed.. Data showed that PTX-11 triggered a remarkable depolymerizing effect on actin cytoskeleton and also modifications in the shape of cells. In contrast, PTX-2SA did not evidence the same effects.. Our findings point out that (i) the actin cytoskeleton is a common target for PTX-11, PTX-2 and PTX-1, but not for PTX-2SA, and (ii) this difference in activity is related to the presence or absence of an intact lactone ring in their structures.

Topics: Actins; Cell Line, Tumor; Cytoskeletal Proteins; Cytoskeleton; Furans; Humans; Lactones; Macrolides; Marine Toxins; Models, Molecular; Neuroblastoma; Pyrans

The stereocontrolled synthesis of the C1-C16 ABC spiroacetal-containing tricyclic fragment of pectenotoxin-7 6 has been accomplished. The key AB spiroacetal aldehyde 9 was successfully synthesized via acid catalyzed cyclization of protected ketone precursor 28 that was readily prepared from aldehyde 12 and sulfone 13. The syn stereochemistry in aldehyde 12 was installed using an asymmetric aldol reaction proceeding via a titanium enolate. The stereogenic centre in sulfone 13 was derived from (R)-(+)-glycidol. The absolute stereochemistry of the final spiroacetal aldehyde 9 was confirmed by NOE studies establishing the (S)-stereochemistry of the spiroacetal centre. Construction of the tetrahydrofuran C ring system began with Wittig olefination of the AB spiroacetal aldehyde 9 with (carbethoxyethylidene)triphenylphosphorane 10 affording the desired (E)-olefin 32. Appendage of a three carbon chain to the AB spiroacetal fragment was achieved via addition of acetylene 11 to the unstable allylic iodide 39. Epoxidation of (E)-enyne 8 via in situ formation of L-fructose derived dioxirane generated the desired syn-epoxide 36. Semi-hydrogenation of the resulting epoxide 36 followed by dihydroxylation of the alkene effected concomitant cyclization, thus completing the synthesis of the ABC spiroacetal ring fragment 6.

Topics: Epoxy Compounds; Furans; Macrolides; Magnetic Resonance Spectroscopy; Marine Toxins; Models, Molecular; Pyrans; Spiro Compounds

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.

Topics: Animals; Bivalvia; Chromatography, Liquid; Dinoflagellida; Environmental Monitoring; Ethers, Cyclic; Furans; Macrolides; Marine Toxins; Mollusk Venoms; New Zealand; Okadaic Acid; Oxocins; Phytoplankton; Pyrans; Shellfish; Spectrometry, Mass, Electrospray Ionization