Anne-Marie Legrand

The epidemiology of ciguatera fish poisoning

Ciguatera is a toxin-related disease caused by ingestion of a variety of toxic fish living in tropical or subtropical areas. This article aims to look at the epidemiology of the disease, from both the descriptive and analytical points of view, and to discuss them in relation to environmental aspects and socioeconomic impact.

13C NMR Assignments of ciguatoxin by inverse-detected 2d spectroscopy and an explanation of nmr signal broadening

Abstract 13C NMR assignments of ciguatoxin were achieved with 1.1 mg of the sample (1 μmol). 13C-decoupled HMQC and HSQC spectra revealed 1JCH arising from the flexible part of the molecule which yielded no sharp peaks in the 1D 13C NMR spectrum. These data suggest that contiguous 9- and 7-membered rings (rings F, G) give rise to a slow conformational change, which leads to extreme broadening of both 1H and 13C NMR signals.

Light and electron microscopic studies of pathologic changes induced in mice by ciguatoxin poisoning

Acute poisoning induced by ciguatoxin or ciguatoxin-4c in male ICR mice was examined by light and electron microscopy. Target organs were the heart, medulla of adrenal glands, autonomic nerves and penis. There were no significant differences between the toxicity of ciguatoxin and ciguatoxin-4c. Either i.p. injection or oral administration (0.7 micrograms/kg) resulted in marked swelling and focal necrosis of cardiac muscle cells and effusion into the interstitial space of the heart. Degeneration of cells in the medulla of the adrenal glands was also observed. Continuous erection of the penis was observed in about 15% of the mice suffering from ciguatoxicosis. Although severe diarrhea was brought about by the administration of these phycotoxins, no morphological alterations were seen in the mucosa and muscle layers of the small intestine except in autonomic nerve fibers and synapses. Atropine suppressed the symptoms of diarrhea but had no effect on the injury to the cardiac muscle. Reserpine aggravated the clinical signs and pathological findings. Guanethidine and 5-hydroxy dopamine as well as those undergoing bilateral adrenalectomy had no significant effects on the ciguatoxicosis.

Characterization of mice antisera elicited with a ciguatoxin tetracyclic synthetic ring fragment (JKLM) conjugated to carrier proteins.

As a good alternative to the lack of pure ciguatoxin (CTX), conjugates of JKLM ring fragment, a carboxylic derivative of the right-hand tetracyclic terminus portion of CTX-1B (the most potent CTX) with two carrier proteins have been synthesized. Two procedures using different amount of hapten were evaluated: (i) a bulk technique (3-5 mg) via the N-hydroxysuccinimide ester of the carboxylic fragment in the presence of a water-soluble carbodiimide according to the standard method in aqueous buffer, or (ii) a micro-scale technique (300 microg) via the mixed anhydride method performed in a reversed micellar medium. In both cases, bovine serum albumin and ovalbumin were respectively used for immunization of BALB/c mice and antibody screening by a solid phase enzyme-linked immunosorbent assay (ELISA). Using the conjugates obtained through the micro-scale procedure, a long-term immunization schedule appeared to be more efficient to specifically trigger the mice immune system. These antisera titers determined in an end-point titration standard ELISA format were found around 1/128,000 as compared to 1/16,000 obtained in the short-term protocol (immunogen prepared via the bulk procedure). In competitive inhibition ELISA experiments, both types of antisera did not significantly cross-react with a brevetoxin congener (PbTx-3), okadaic acid (OA), monensin or other polyether compounds, but only sera from the short-term protocol did show high cross-reactivity to CTX-1B (133%). With sera from the long-term protocol, a lower detection limit for JKLM (1.23 x 10(-9) M) was achieved by implementation of a biotin-avidin amplification system rather than by miniaturization of the assay in Terasaki plates. This study confirms the feasibility of the immunological approach for CTXs assay in fish tissues, but also emphasizes the importance of (i) the choice of the hapten to construct a relevant well-defined immunogen, (ii) the immunization schedule to obtain hapten-specific Abs still exhibiting high cross-reactivity to CTXs.

INTRASPECIFIC VARIATION IN THE DINOFLAGELLATE GAMBIERDIZSCUS TOXICUS (DINOPHYCEAE). I. ISOZYME ANALYSIS

Intraspecific variation among 19 isolates of the ciguatera‐causing dinoflagellate Gambierdiscus toxicus Adachi & Fukuyo (Dinophyceae) collected from French Polynesia, New Caledonia, and the French West Indies was investigated by isozyme analysis. Comparison of their cell sizes and growth rates revealed that significant variation exists among these clones. Comparison of electrophoretic patterns for seven enzyme systems indicated that G. toxicus is comprised of numerous biochemically distinct strains. Isolates from Tubuai and Hao appeared to be the most distantly related. Tahitian strains of G. toxicus also showed a remarkably low degree of similarity with the Tubuai isolates. The latter, which were taken from the same locale in Tubuai, also exhibited highly heterogeneous electrophoretic Profiles when compared to each other, suggesting a multiclonal origin. The single isolate analyzed from the Atlantic Ocean was most closely related to Tahitian isolates, despite their geographic separation. Finally, no clear relationship was found between the electrophoretic profiles of these isolates and their capacity to produce ciguatoxic compounds.

Effect of maitotoxin on calcium current and background inward current in isolated ventricular rat myocytes

Maitotoxin (MTX) irreversibly suppressed the voltage-dependent calcium current after a variable delay, an effect which was preceded, in 61% of the cells, by a transient increase in calcium current partly attributable to a shift (4-7 mV) of the activation curve towards negative potentials. MTX also induced the development of a voltage-independent background inward current which did not occur in the absence of external calcium and was reduced by removal of external sodium, by calcium channel blockers and by high concentrations of quinidine. MTX-induced single channel activity consisted of long lasting bursts of inward current. Channel activity was voltage-independent, with a unitary conductance of 14 pS and an extrapolated reversal potential of +16 mV. Single-channel current amplitude was not detectably reduced in the absence of external calcium but strongly reduced in the absence of external sodium, in the presence of 2 mM nickel or when external sodium was replaced by 96 mM calcium or 50 mM barium. The channel activity was also inhibited by quinidine. It is concluded that MTX alters, then suppresses the voltage-activated calcium current and induces the development of a voltage-independent inward current, part of which results from the opening of nickel-sensitive cation channels, mostly permeable to sodium ions.