In vivo subchronic effects of ciguatoxin-related compounds, reevaluation of their toxicity.

Ciguatoxins are marine compounds that share a ladder-shaped polyether structure produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa, and include maitotoxins (MTX1 and MTX3), ciguatoxins (CTX3C) and analogues (gambierone), components of one of the most frequent human foodborne illness diseases known as ciguatera fish poisoning. This disease was previously found primarily in tropical and subtropical areas but nowadays, the dinoflagellates producers of ciguatoxins had spread to European coasts. One decade ago, the European Food Safety Authority has raised the need to complete the toxicological available data for the ciguatoxin group of compounds. Thus, in this work, the in vivo effects of ciguatoxin-related compounds have been investigated using internationally adopted guidelines for the testing of chemicals. Intraperitoneal acute toxicity was tested for maitotoxin 1 at doses between 200 and 3200 ng/kg and the acute oral toxicity of Pacific Ciguatoxin CTX3C at 330 and 1050 ng/kg and maitotoxin 1 at 800 ng/kg were also evaluated showing not effects on mice survival after a 96 h observation period. Therefore, for the following experiments the oral subchronic doses were between 172 and 1760 ng/kg for gambierone, 10 and 102 ng/kg for Pacific Ciguatoxin CTX3C, 550 and 1760 ng/kg for maitotoxin 3 and 800, 2560 and 5000 ng/kg for maitotoxin 1. The results presented here raise the need to reevaluate the in vivo activity of these agents. Although the intraperitoneal lethal dose of maitotoxin 1 is assumed to be 50 ng/kg, without chemical purity identifications and description of the bioassay procedures, in this work, an intraperitoneal lethal dose of 1107 ng/kg was obtained. Therefore, the data presented here highlight the need to use a common procedure and certified reference material to clearly establish the levels of these environmental contaminants in food.

Oral Chronic Toxicity of the Safe Tetrodotoxin Dose Proposed by the European Food Safety Authority and Its Additive Effect with Saxitoxin.

Tetrodotoxin (TTX) is a potent natural toxin causative of human food intoxications that shares its mechanism of action with the paralytic shellfish toxin saxitoxin (STX). Both toxins act as potent blockers of voltage-gated sodium channels. Although human intoxications by TTX were initially described in Japan, nowadays increasing concern about the regulation of this toxin in Europe has emerged due to its detection in fish and mollusks captured in European waters. Currently, TTX is only regularly monitored in Dutch fishery products. However, the European Food Safety Authority (EFSA) has established a safety level of 44 µg/kg TTX as the amount of toxin that did not cause adverse effects in humans. This level was extrapolated considering initial data on its acute oral toxicity and EFSA remarked the need for chronic toxicity studies to further reduce the uncertainty of future toxin regulations. Thus, in this work, we evaluated the oral chronic toxicity of TTX using the safety levels initially recommended by EFSA in order to exclude potential human health risks associated with the worldwide expanding presence of TTX. Using internationally recommended guidelines for the assessment of oral chronic toxicity, the data provided here support the proposed safety level for TTX as low enough to prevent human adverse effects of TTX even after chronic daily exposure to the toxin. However, the combination of TTX with STX at doses above the maximal exposure level of 5.3 µg/kg body weight derived by EFSA increased the lethality of TTX, thus confirming that both TTX and paralytic shellfish toxins should be taken into account to assess human health risks.

Chronic In Vivo Effects of Repeated Exposure to Low Oral Doses of Tetrodotoxin: Preliminary Evidence of Nephrotoxicity and Cardiotoxicity.

Tetrodotoxin (TTX) is one of the most potent naturally occurring neurotoxins. InitiallyTTX was associated with human food intoxications in Japan, but nowadays, concerns about thehuman health risks posed by TTX have increased in Europe after the identification of the toxin infish, marine gastropods, and bivalves captured in European waters. Even when TTX monitoring isnot currently performed in Europe, an acute oral no observable effect level (NOAEL) of 75 µg/kghas been recently established but, to date, no studies evaluating the chronic oral toxicity of TTXhave been released, even when EFSA has highlighted the need for them. Thus, in this work, thechronic effects of low oral TTX doses (below the acute lethal dose 50) were evaluated followinginternationally adopted guidelines. The results presented here demonstrate that low oral doses ofTTX have deleterious effects on renal and cardiac tissues. Moreover, alterations in bloodbiochemistry parameters, urine production, and urinalysis data were already detected at the oraldose of 75 µg/kg after the 28 days exposure. Thus, the data presented here constitute an initialapproach for the chronic evaluation of the in vivo toxicity of tetrodotoxin after its ingestion throughcontaminated fishery products.

A fluorimetric microplate assay for detection and quantitation of toxins causing paralytic shellfish poisoning.

Paralytic shellfish poisoning is one of the most severe forms of food poisoning. The toxins responsible for this type of poisoning are metabolic products of dinoflagellates, which block neuronal transmission by binding to the voltage-gated Na(+) channel. Accumulation of paralytic toxins in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread and thorough monitoring program for mollusk toxicity. All of these programs require periodical collection and analysis of a wide range of shellfish. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would streamline this process. Our laboratory has developed a fluorimetric microplate bioassay that rapidly and specifically determines the presence of paralytic shellfish toxins in many seafood samples. This method is based on the pharmacological activity of toxins and involves several steps: (i) Incubation of excitable cells in 96 well microtiter plates with the fluorescent dye, bis-oxonol, the distribution of which across the membrane is potential-dependent. (ii) Cell depolarization with veratridine, a sodium channel-activating toxin. (iii) Dose-dependent inhibition of depolarization with saxitoxin or natural samples containing paralytic shellfish toxins. Measuring toxin-induced changes in membrane potential allowed for quantification and estimation of the toxic potency of the samples. This new approach offers significant advantages over classical methods and can be easily automated.