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

Abstract

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.

Figure 1

Reversed-phase high-performance liquid chromatography (HPLC) plots of (A) the water-soluble neurotoxic extract obtained from mussel samples, using a C18 symmetry column, and (B) the water-soluble neurotoxic fraction, possessing the entire toxic activity, using a C18 GOLD ODS column. Elution was performed with a linear gradient of 20%–60% acetonitrile in acidified water run between 2 and 35 min at a flow rate of 1 mL/min. The column effluent was monitored at 210 nm. The active fraction, determined with the mouse bioassay, was repurified under the same conditions to obtain the final purified product.

Figure 2

The extract ion chromatogram of the ion 228.289 m/z and MS spectra (scan range m/z 200–1800) of C17-SAMT eluted at 32.92 min with a molecular mass of 288.289 (m/z) (B,D) and C17-SPA eluted at 32.80 min (A,C) from LC-MS analysis of water-soluble neurotoxic fraction. (E) MS/MS spectrum of selected ion m/z 288.289. (F) Chemical structure of C17-SPA.

Figure 3

In vivo effects of C17-SAMT on the multimodal excitability properties of mouse neuromuscular system. (A) On-line recordings of the effect of C17-SAMT (25–168 μg/100 μL PBS) injections on the CMAP maximal amplitude registered continuously as a function of time, from tail muscle following caudal motor nerve stimulation. Values are expressed relatively to those before toxin injections. The arrow indicates the time of injections; (B) Traces of CMAPs recorded before and 1 and 8 h after C17-SAMT (50 μg/100 μL PBS) injection. Notice the increased excitability threshold (arrows) after 1 h toxin injection; (C) Dose-response curves of the effects of C17-SAMT (black circles) and C17-SPA (white circles) on the CMAP maximal amplitude. Values represent means ± SD of data obtained from 3 to 4 mice, and are expressed relatively to those obtained before toxin injections. The curves were calculated from typical sigmoid non-linear regression through data points (r² ≥ 0.996). The toxin dose required to block 50% of the CMAP amplitude (IC₅₀) was 50 μg (C17-SAMT) and 108 μg (C17-SPA)/100 μL PBS.

Figure 4

Excitability waveforms recorded, in vivo, from tail muscle following caudal motor nerve stimulation in 10 months-old mice before (black circles) and 1 (white circles) and 8 h (grey circles) after injection of 100 μL PBS solution containing 50 μg C17-SAMT. (C1) Current-threshold relationships; (C2) Charge-duration relationships established from strength-duration testing; (C3) Threshold electrotonus; and (C4) Recovery cycle. Mean ± SD of data obtained from four different mice.

Figure 5

Effect of C17-SAMT on directly-elicited isometric twitch and tetanic contractions of an isolated mouse hemidiaphragm. Representative single twitch (A) and tetanic contraction recordings (D, 40 Hz) under control conditions, and in the presence of 65 μg/mL C17-SAMT (B, single twitch) and (E, 40 Hz tetanic contraction). Note the marked block of the twitch and tetanic contractions induced by the toxin (B,E). (C,F) Reversal of C17-SAMT effect following 50 min wash out of C17-SAMT from the medium. Vertical calibration in (A) applies to all recordings.

Figure 6

The time-dependent effect of C17 SAMT (65 μg/mL) on twitch (open circles) and tetanic tension amplitude (filled circles) evoked by direct muscle stimulation (in % relative to control). The arrow-head indicates the addition of C17 SAMT to the medium, and the arrow the start of the washout of the toxin from the medium.

Figure 7

Microscopic observation of Aspergillus sp. strain obtained from contaminated mussels.