domoic-acid and palytoxin

At the European level, detection of marine neurotoxins in seafood is still based on ethically debated and expensive in vivo rodent bioassays. The development of alternative methodologies for the detection of marine neurotoxins is therefore of utmost importance. We therefore investigated whether and to what extent a multielectrode array (MEA) approach can be used as an in vitro alternative for screening of marine neurotoxins potentially present in seafood.. This MEA approach utilizes rat cortical neurons comprising a wide range of ion channels/pumps and neurotransmitter receptors targeted by marine neurotoxins. We tested the effects of neurotoxic model compounds, pure marine neurotoxins, and extracts from contaminated seafood on neuronal activity of rat cortical neurons cultured on commercial 48-well plates to increase throughput.. We demonstrate that the MEA approach has a sensitivity of 88% (7/9 model compounds, 6/6 pure marine neurotoxins, and 2/2 marine neurotoxins present in seafood extracts were correctly identified) and a good reproducibility compared to existing in vitro alternatives. We therefore conclude that this MEA-based approach could be a valuable tool for future food safety testing.

Topics: Acrylamides; Animal Use Alternatives; Animals; Cells, Cultured; Ciguatoxins; Cnidarian Venoms; Fishes; Food Contamination; Food Safety; Kainic Acid; Marine Toxins; Neurons; Neurotoxins; Oxocins; Rats; Rats, Wistar; Reproducibility of Results; Seafood; Tetrodotoxin

Dysfunction or deficiency of the Na(+)/K(+)-ATPase appears to be a common event in a variety of pathological conditions in the central nervous system. Studies on neurotoxicity associated to impaired Na(+)/K(+)-ATPase activity have focused on NMDA receptors, while the involvement of non-NMDA receptors has been much less explored. We show that mild, non-toxic, exposures to the Na(+)/K(+)-ATPase inhibitor palytoxin (PTX) synergistically sensitized the vulnerability of neurons to normally non-toxic concentrations of domoic acid, leaving NMDA receptor-mediated excitotoxic response unaltered. Enhancement of excitotoxicity required at least 1 h pre-exposure to PTX, was not observed after longer exposures to PTX, and did not require RNA synthesis. PTX caused a voltage-sensitive Na(+) channel-independent increase in intracellular Na(+). Both intracellular Na(+) increase and potentiation of excitotoxicity depended upon the external concentrations of Na(+) and Cl(-), and were suppressed by the anion exchanger blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid in a dose-dependent manner. Other stilbene derivatives, chloride channel antagonists or Na(+) cotransporter inhibitors proved ineffective. Our results demonstrate a crucial role for Na(+)/K(+)-ATPase activity in determining neuronal vulnerability to domoic acid-mediated excitotoxicity. They also raise reasonable concern about possible risks for human health associated to the ingestion of low amounts of phycotoxins PTX and domoic acid in food.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acrylamides; Animals; Calcium; Calcium Channel Blockers; Cell Survival; Cells, Cultured; Cerebellum; Cnidarian Venoms; Drug Synergism; Extracellular Space; Intracellular Space; Kainic Acid; Neurons; Neurotoxins; Rats; RNA; Sodium; Sodium-Potassium-Exchanging ATPase; Time Factors

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Topics: Acrylamides; Animals; Cell Line, Tumor; Cnidarian Venoms; Dose-Response Relationship, Drug; Furans; Glioma; Hybrid Cells; Kainic Acid; Macrolides; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxocins; Pyrans; Saxitoxin; Time Factors; Toxicity Tests

The monoclonal antibody to ciguatoxin (CTX) produced from a hybridoma cell line was assayed for the detection of four congeners of CTX: Pacific ciguatoxin-1 (P-CTX-1), Pacific ciguatoxin-2 (P-CTX-2), Pacific ciguatoxin-3 (P-CTX-3), and Caribbean ciguatoxin-1 (C-CTX-1) and related marine toxins, including domoic acid, palytoxin, and okadaic acid, using a modified enzyme-linked immunosorbent assay (ELISA). Lower detection limits were assessed and linearity was statistically established (P<0.05) for P-CTX-1, P-CTX-2, and P-CTX-3 and C-CTX-1 at concentrations ranging from 0 to 5.00 ng, while the other marine toxins showed statistically insignificant cross-reactivities at similar concentrations. Thus, the monoclonal antibody to CTX is able to specifically detect various CTX congeners at levels comparable to those naturally occurring in ciguatoxic fish.

Topics: Acrylamides; Antibodies, Monoclonal; Caribbean Region; Ciguatera Poisoning; Ciguatoxins; Cnidarian Venoms; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Kainic Acid; Okadaic Acid; Pacific Ocean; Seafood