An Evaluation of the Cytotoxic and Genotoxic Effects of the Marine Toxin C17-SAMT in Human TK6 and HepaRG Cell Lines.

This study investigates the genotoxicity and cytotoxicity of C17-sphinganine analog mycotoxin (C17-SAMT) using in vitro assays. C17-SAMT was previously identified as the cause of unusual toxicity in cultured mussels from the Bizerte Lagoon in northern Tunisia. While a previous in vivo genotoxicity study was inconclusive, in vitro results demonstrated that C17-SAMT induced an increase in micronucleus formation in human lymphoblastoid TK6 cells at concentrations of 0.87 µM and 1.74 µM. In addition, multiparametric cytotoxicity assays were performed in the human hepatoma HepaRG cell line, which showed that C17-SAMT induced mitochondrial dysfunction, decreased cellular ATP levels, and altered the expression of various proteins, including superoxide dismutase SOD2, heme oxygenase HO-1, and NF-κB. These results suggest that C17-SAMT is mutagenic in vitro and can induce mitochondrial dysfunction in HepaRG cells. However, the exact mode of action of this toxin requires further investigation. Overall, this study highlights the potential toxicity of C17-SAMT and the need for further research to better understand its effects.

Marine toxin C17-SAMT causes major structural damage to vital organs in mice following subchronic toxicity trials.

C17-sphinganine analog mycotoxin (C17-SAMT) has been characterized as the contaminant responsible for the atypical toxicity reported in mussels from the Bizerte lagoon (northern Tunisia) over the past decade. C17-SAMT exhibited common symptoms of toxicity in mice, including flaccid paralysis and severe respiratory distress, followed by rapid death. To determine the potential health risks of this neurotoxin, we assessed its subchronic toxicity according to the recommendations of OCDE n° 407. The body weight and the structural changes of vital organs were recorded. Biochemical and hematological parameters were also quantified. Macroscopic observations showed that mice treated with 0.9, 9, and 90 µg/kg C17-SAMT had significantly reduced stomach weights, swollen and fragile intestines, and signs of nephritis with renal abscesses. Transaminase assays pointed out that exposure to C17-SAMT can lead to transaminitis. Above-average lactate dehydrogenase values were recorded in both the treated and satellite groups. Hematology data showed a significant reduction in red blood cell counts in high-dose-treated group. Reductions in hemoglobin and hematocrit were also recorded. Mean leukocyte counts were significantly elevated in the high-, mid-dose treated and satellite groups. At the microscopic level, we noted myocardial atrophy and hyperemia. In the lungs, we noted necrosis associated with macrophages perivascular infiltration and congestion. The kidneys showed mild inflammation and glomerular atrophy. The stomach exhibited mucosal atrophy, while a thin colon and distended small intestine were observed in high-dose-treated group. The liver was affected by vascular congestion, inflammatory infiltration, and lobular necrosis that evolved into acute hepatitis. Lesions, such as inflammatory infiltration and mild necrosis of the liver, cortical abscess with central necrosis in the kidney, and mild congestion of cardiac tissue were recorded in the satellite group.

Investigation of the Genotoxic Potential of the Marine Toxin C17-SAMT Using the In Vivo Comet and Micronucleus Assays.

The contaminant responsible for the atypical toxicity reported in mussels from Bizerte Lagoon (Northern Tunisia) during the last decade has been characterized as C17-sphinganine analog mycotoxin (C17-SAMT). This neurotoxin showed common mouse toxic symptoms, including flaccid paralysis and severe dyspnea, followed by rapid death. For hazard assessment on human health, in this work we aimed to evaluate the in vivo genotoxic effects of this marine biotoxin using the classical alkaline and modified Fpg comet assays performed to detect DNA breaks and alkali-labile sites as well as oxidized bases. The micronucleus assay was used on bone marrow to detect chromosome and genome damage. C17-SAMT induces a statistically insignificant increase in DNA tail intensity at all doses in the duodenum, and in the spleen contrary to the liver, the percentage of tail DNA increased significantly at the mid dose of 300 µg/kg b.w/d. C17-SAMT did not affect the number of micronuclei in the bone marrow. Microscopic observations of the liver showed an increase in the number of mitosis and hepatocytes' cytoplasm clarification. At this level of study, we confirm that C17-SAMT induced DNA damage in the liver but there was no evidence of effects causing DNA oxidation or chromosome and genome damage.

Purification, Toxicity and Functional Characterization of a New Proteinaceous Mussel Biotoxin from Bizerte Lagoon.

The marine environment is known to be occupied by microorganisms. The potential toxicity of some of these marine microorganisms, that are capable of producing unknown biotoxins, has always been underestimated. Indeed, these biotoxins may be a threat to human health through the consumption of contaminated seafood and fish. For more than ten years, recurrent but atypical toxicity has been detected in mussels from Bizerte lagoon (North of Tunisia) during routine tests. In this study, we have isolated and characterized a new proteinaceous marine biotoxin, named Mussel Toxic Peptide (MTP). Using HPLC, electrophoresis and LC/MS studies, we showed that MTP has a protein characteristic UV-spectrum, can be visualized by protein specific reagents such as Coomassie, and has a molecular mass of 6.4 kDa. Patch-clamp experiments performed on cultured N18 neuroblastoma cells revealed that MTP (0.9-18 µM) markedly inhibited voltage-gated Na current, but was about 23 times less active in blocking voltage-gated K current at equimolar concentrations. To the best of our knowledge, this is the first time that a proteinaceous marine biotoxin with relatively high molecular mass is isolated and involved in the contamination of mussels harvested from shellfish farming areas.

Toxic c17-sphinganine analogue mycotoxin, contaminating tunisian mussels, causes flaccid paralysis in rodents.

Severe toxicity was detected in mussels from Bizerte Lagoon (Northern Tunisia) using routine mouse bioassays for detecting diarrheic and paralytic toxins not associated to classical phytoplankton blooming. The atypical toxicity was characterized by rapid mouse death. The aim of the present work was to understand the basis of such toxicity. Bioassay-guided chromatographic separation and mass spectrometry were used to detect and characterize the fraction responsible for mussels' toxicity. Only a C17-sphinganine analog mycotoxin (C17-SAMT), with a molecular mass of 287.289 Da, was found in contaminated shellfish. The doses of C17-SAMT that were lethal to 50% of mice were 750 and 150 µg/kg following intraperitoneal and intracerebroventricular injections, respectively, and 900 µg/kg following oral administration. The macroscopic general aspect of cultures and the morphological characteristics of the strains isolated from mussels revealed that the toxicity episodes were associated to the presence of marine microfungi (Fusarium sp., Aspergillus sp. and Trichoderma sp.) in contaminated samples. The major in vivo effect of C17-SAMT on the mouse neuromuscular system was a dose- and time-dependent decrease of compound muscle action potential amplitude and an increased excitability threshold. In vitro, C17-SAMT caused a dose- and time-dependent block of directly- and indirectly-elicited isometric contraction of isolated mouse hemidiaphragms.

Analysis of the action of gymnodimine-A and 13-desmethyl spirolide C on the mouse neuromuscular system in vivo.

Gymnodimine-A and 13-desmethyl spirolide C are marine toxins belonging to the cyclic imine group produced by Karenia selliformis and Alexandrium ostenfeldii/peruvianum dinoflagellates, respectively. The aim of this work was to analyze the pharmacological properties of both toxins (at sub-lethal doses) on neuromuscular excitability, when injected locally to isoflurane-anesthetized mice, using a multimodal minimally-invasive in vivo electrophysiological approach. The main effect of both toxins was a marked reversible time- and dose-dependent decrease of the compound muscle action potential recorded from the tail muscle in response to caudal motor nerve stimulation. The dose-response curves of gymnodimine-A and 13-desmethyl spirolide C effect on the maximal amplitude of compound muscle action potential revealed 50% inhibitory doses of 51 ng/mouse (i.e. 1.6 µg/kg or 3.3 nmol/kg mouse) and 0.18 ng/mouse (i.e. 6 ng/kg or 0.01 nmol/kg mouse), respectively. The blocking effect occurred without significant modification of motor nerve excitability parameters. It is concluded that the inhibition of the mouse compound muscle action potential, induced by gymnodimine-A and 13-desmethyl spirolide C, results from an action of these toxins at the level of the skeletal neuromuscular junction, since both cyclic imine toxins are known to interact and block muscle-type nicotinic acetylcholine receptors. In the present in vivo study, 13-desmethyl spirolide C was about 300 fold more active than gymnodimine-A on equimolar basis. The present in vivo approach, associated to recent progress in chemical synthesis of cyclic imine toxins, paves the way for more detailed structure-activity studies to obtain new and more potent synthetic analogs.

Quantitative determination of gymnodimine-A by high performance liquid chromatography in contaminated clams from Tunisia coastline.

Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for extraction, detection and quantification of GYM-A in toxic clams. The extraction of GYM-A from the digestive gland of clams in acetone, subsequent clean-up with diethyl ether and extraction with dichloromethane is the more valid protocol. Chromatography analyses were performed using a gradient of acetonitrile-water (10:90 to 90:10), containing trifluoroacetic acid (0.1%) for 20 min at 1 mL/min rate with a C18 column. Recovery rates exceeded 96%, and limits of detection and quantification were 5 ng/mL and 8 ng/g digestive gland, respectively. Repeatability and reproducibility were tested for various samples containing different levels of GYM-A. A significant correlation was observed between toxicity level of samples and the determined amount of GYM-A. Also, the persistence of GYM-A in contaminated clams from Boughrara lagoon was demonstrated. The kinetics discharge study of GYM-A in controlled medium, during 1 month, showed that the process of depuration was biphasic with an exponential discharge of 75% of the total amount of sequestered GYM-A during the first 12 days followed by a slow discharge (>10%) for the subsequent days up to the seventeenth day. This is the first time that a quantitative study of GYM-A in clams from Tunisian coasts is performed through the development of a new method for detection and quantify of this phycotoxin. We found HPLC-UV a reliable and suitable alternative to the mouse bioassay.

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.