Domoic Acids Research Articles

Cytotoxicity and intestinal permeability of phycotoxins assessed by the human Caco-2 cell model

Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.

Determination of Cyanotoxins and Phycotoxins in Seawater and Algae-Based Food Supplements Using Ionic Liquids and Liquid Chromatography with Time-Of-Flight Mass Spectrometry.

An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid–liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 18.3 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample.

Insecticidal activity guided isolation of palytoxin from a red alga, Chondria armata

A red alga, Chondria armata, was used as a vermifuge in Japan. Previously, we isolated domoic acids from C. armata guided by the insecticidal activity against American cockroaches. Further investigations led to the discovery of a compound one-thousand times more toxic than domoic acid. It showed lethal activity at 5ng against cockroaches. The insecticidal compound was identified as palytoxin by means of NMR and MS. Careful interpretation of the NMR spectra allowed us to refine the previously reported NMR assignment of palytoxin. Palytoxin has been isolated from various organisms, and it is significant that palytoxin was isolated from marine plants.

Toxin production and temperature-induced morphological variation of the diatom Pseudo-nitzschia seriata from the Arctic

This is the first record of a domoic acid-producing organism from polar waters. Strains of the cold-water diatom Pseudo-nitzschia seriata were isolated from water samples collected from the west coast of Greenland. Growth experiments on two of the strains showed similar growth rates at 4 °C and production of DA (domoic acid) and the two isomers IA (isodomoic acid A) and IB (isodomoic acid B). Both strains produced significantly higher amounts of DA than of IA and IB. The concentrations of domoic acids were 1.46–1.93 pg per cell, and thus have the potential to pose a risk for amnesic shellfish poisoning. Four strains of P. seriata were cultured at different temperatures (4, 10 and 15 °C), and morphological differences were found at the different temperatures. Similar differences have been observed previously in field samples from different geographical areas and have resulted in uncertainty regarding the taxonomy of P. seriata and related species. Transmission electron microscopy showed that at 4 °C the strains corresponded to the original description of P. seriata. At higher temperatures, the number of rows of poroids in the striae as well as the density of poroids decreased, and cells then resembled the closely related P. australis. The Greenlandic strains of P. seriata did not grow well at 15 °C. The identity of the strains was confirmed by analyses of ITS1, 5.8S and ITS2 rDNA from P. seriata and P. australis, using strains from different geographical areas. There was a close phylogenetic relationship between P. seriata and P. australis but the analyses demonstrated consistent molecular differences between the two species, in spite of the morphological similarity.

Photostability of Kainic Acid in Seawater

The environmental degradation of a mixture of domoic acid (DA) and kainic acid (KA) in seawater with and without added transition metals is reported. The association constants for kainic acid with Fe (III) and Cu (II) were determined using (1)H nuclear magnetic resonance (NMR; K1,Fe(III) = 2.27 x 10(12), K2,Fe(III) = 8.99 x 10(8), K1,Cu(II) = 1.38 x 10(10), and K2,Cu(II) = 4.35 x 10(7)). The photochemical half-life of kainic acid has been determined to be significantly longer (40-100 h) than that of domoic acid in corresponding marine systems (12-34 h). The significance of this finding was highlighted by a comparison of the quantification of a mixture of kainic and domoic acids during photodegradation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and the widely used competitive enzyme-linked immunosorbent assay (cELISA; Biosense Laboratories) method. The MS-based analysis showed that approximately 50% of the DA was photodegraded within 15 h. In contrast, the domoic acid cELISA assay reported that the concentration essentially remained unchanged over this period. The possibility of interference from naturally occurring kainic acid during cELISA measurements could lead to the overestimation of total domoic acid, especially if they occur in mixtures in sunlit waters.

Committee on Natural Toxins and Food Allergens: Marine and Freshwater Toxins

Committee on Natural Toxins and Food Allergens: Marine and Freshwater Toxins

Crystal Structures of the GluR5 and GluR6 Ligand Binding Cores: Molecular Mechanisms Underlying Kainate Receptor Selectivity

Little is known about the molecular mechanisms underlying differences in the ligand binding properties of AMPA, kainate, and NMDA subtype glutamate receptors. Crystal structures of the GluR5 and GluR6 kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate have now been solved. The structures reveal that the ligand binding cavities are 40% (GluR5) and 16% (GluR6) larger than for GluR2. The binding of AMPA- and GluR5-selective agonists to GluR6 is prevented by steric occlusion, which also interferes with the high-affinity binding of 2S,4R-4-methylglutamate to AMPA receptors. Strikingly, the extent of domain closure produced by the GluR6 partial agonist kainate is only 3 degrees less than for glutamate and 11 degrees greater than for the GluR2 kainate complex. This, together with extensive interdomain contacts between domains 1 and 2 of GluR5 and GluR6, absent from AMPA receptors, likely contributes to the high stability of GluR5 and GluR6 kainate complexes.

Central non-NMDA receptors of a honey bee with hereditary kynurenine deficiency.

Pharmacological characteristics of non-NMDA receptors involved in associative learning were studied in wild honey bees (normal) and carriers of snow laranja mutation (kynurenine deficiency) by pharmacological analysis and behavioral criteria. The effects of systemic injections of non-NMDA receptor agonists (AMPA, kainic, quisqualic, and domoic acids), AMPA receptor antagonist (NS257-HCl), and AMPA receptor modulator (cyclothiaside) on retention of conditioned reflexes in short-term memory (1 min after the end of learning) were studied. The pharmacological characteristics of non-NMDA receptors were changed in snow laranja mutants with kynurenine deficiency.

Kainic and domoic acids differentially affect NADPH-diaphorase neurons in the mouse hippocampal formation

We investigated changes in numbers of nitric-oxide-producing cells in the hippocampal formation, striatum, and temporal cortex of mice 24 h after intraperitoneal administration of kainic acid (5, 10, 15, and 20 mg/kg) or domoic acid (1, 2, and 4 mg/kg). Nitric-oxide-producing cells were demonstrated histochemically by staining for nicotinamide adenine dinucleotide phosphate diaphorase. Nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the dentate gyrus and the subiculum did not change in number following administration of kainic acid or domoic acid at any dose. Positive neurons in the CA3 region of mice treated with kainic acid or domoic acid at any dose were significantly fewer than in controls. Although the numbers of positive neurons in the CA1/CA2 regions did not differ from those of controls at any of the four doses of kainic acid, positive cells in the CA1/CA2 were significantly more numerous than in controls at any dose of domoic acid. Although no significant differences in the numbers of positive neurons in the striatum were apparent between controls and any of the four doses of kainic acid, domoic acid significantly decreased the numbers of such cells. These results suggest that systemically administered kainic acid and domoic acid affect differentially nitric-oxide-producing cells in the hippocampal formation.

Domoic and quisqualic acids as potent amino acid excitants of frog and rat spinal neurones.

THE finding that kainic acid (Fig. 1a) is a potent glutamate-like excitant of rat cerebral1 and cat spinal2 neurones has led us to test domoic acid (Fig. 1b), which is structurally related to kainic acid and wnich also occurs in certain marine algae and has similar anthelmintic properties3,4. We have also compared the actions of these two compounds with those of α-allo-kainic acid (Fig. 1c), a stereo-isomer of kainic acid and a weaker excitant of rat cerebral neurones1, and with quisqualic acid (Fig. 1d), an ascaricidal compound isolated from seeds of the plant genus Quisqualis5. Quisqualic acid is highly potent as a glutamate agonist at the crayfish neuromuscular junction6. Compounds were tested on rat spinal interneurones by micro-electrophoresis7 and on frog spinal motoneurones by superfusion of the procaine-blocked8 hemisected spinal cord in vitro9. On both these preparations (see Table 1), the anions of domoic and quisqualic acids were found to be at least two orders of magnitude more potent than L-glutamate, and equal to or stronger than kainate, whereas α-allo-kainate was a considerably weaker excitant.