Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation

Abstract

Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate receptor binding assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by receptor binding-assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine receptors.

Keywords: 28-o-palmitoyl ester of pinnatoxin-G; Cyclic imine toxins; Microplate receptor-binding assay; Nicotinic acetylcholine receptors; Pinnatoxin-G; Portimine-A; Two-electrode voltage clamp electrophysiology.

FIGURE 1

1A. Microplate receptor-binding assay showing the inhibition of biotin-α-bungarotoxin by digestive-gland extracts from mussels collected in Ingril lagoon. DG-samples were analyzed by sextuplicate. Plate signal (C-P): control wells processed without membrane coating. 100% signal (C-MB): control wells in which Torpedo-nAChRs were incubated in the absence of toxin or extract samples. 100% inhibition (C-INH): control wells in which Torpedo-nAChRs were incubated with 1 × 10⁻⁶ M α-BgTx. 1B. Inhibition binding potency of digestive-gland extracts from mussels collected in Ingril lagoon. OD₄₉₂ data were expressed in inhibition percent using Equation 1 (Material and methods). Data are mean values ± SEM of sextuplicate assays of at least two independent experiments. 1C. Inhibition binding potency of shellfish extracts provided by Agri-food and Biosciences Institute (AFBI). OD492 data were expressed in inhibition percent using Equation 1 (Material and methods). Data are mean values ± SEM of sextuplicate assays of at least two independent experiments. 1D. UPLC-MS/MS chromatogram showing the simultaneous analysis of seven cyclic imine toxins and 28-O-palmitoyl ester of pinnatoxin-G. The MRM conditions, retention time and MS-signal are indicated in the figure. 1E. Calibration curves for seven cyclic imine toxins and 28-O-palmitoyl ester of pinnatoxin-G. 5 μl of a toxin standard mix in the concentration range of 1 pM to 1 μM was loaded to the BEH C18 column (see Material and Methods). Toxin quantification was done by integration of the MRM peak area of each analyte. Abbreviations: DG: digestive glands AFBI: Shellfish samples provided by Agri-food and Biosciences Institute; PORT-A: Portimine-A; GYM-A: gymnodimine-A; 13,19-SPX-C: 13,19-didesmethyl-spirolide-C; PnTX-A: pinnatoxin-A; 13-SPX-C: 13-desmethyl spirolide-C; 20-Me-SPX-G: 20-methyl spirolide-G; PnTX-G: Pinnatoxin-G; Palmitoyl-PnTX-G: 28-O-palmitoyl pinnatoxin-G.

FIGURE 2

2A. NMR spectrum of portimine-A (inset). NMR was performed at 20°C on a Bruker DRX700 spectrometer equipped with a triple resonance cryoprobe. 2B. MS/MS spectrum of portimine-A showing its fragmentation pattern at the collision-induced dissociation condition for MRM. 2C. UPLC-MS/MS chromatogram showing the simultaneous detection of portimine-A (PORT-A), gymnodimine-A (GYM-A), pinnatoxin-A (PnTX-A), 13-desmethyl spirolide-C (13-SPX-C) pinnatoxin-G (PnTX-G) and 28-O-palmitoyl pinnatoxin-G (Palmitoyl-PnTX-G) in the digestive gland extract sample 13/414-2 collected in Ingril Lagoon (South France). The MRM conditions, retention time and MS-signal are indicated in the figure. Abbreviations: m/z: mass-to-charge ratio.

FIGURE 3

3A. Chemical structure of pinnatoxin-A, pinnatoxin-G and 28-O-palmitoyl pinnatoxin-G; R = ester 16:0. 3B Mass spectrum of 28-O-palmitoyl ester of pinnatoxin-G showing its fragmentation pattern at the collision-induced dissociation condition for MRM. 3C. Inhibition binding potency of 28-O-palmitoyl pinnatoxin-G compared to its precursor molecule pinnatoxin-G and to pinnatoxin-A. Each point represents the mean value ± SEM of three independent experiments using the Microplate-RBAssay. D. Antagonistic effect of 2.5 μM 28-O-palmitoyl ester of pinnatoxin-G on Torpedo-nAChR of muscle embryonic type recorded using two-electrode voltage clamp electrophysiology. The perfusion protocol was as follows: A clamped oocyte at a holding potential of −60 mV was perfused with 25 μM ACh (ACh) for 15 s at 150 s intervals. Thereafter, the oocyte was perfused with 2.5 μM 28-O-palmitoyl ester of pinnatoxin-G (Pal) for 45 s and immediately after it was exposed to a mixture of 25 μM ACh and 2.5 μM 28-O-palmitoyl ester of pinnatoxin-G (Mix). The oocyte was washed with Ringer-Ba and pulses of 25 μM ACh were recorded twice at 150s intervals. E. Concentration-dependent antagonistic effect of 28-O-palmitoyl ester of pinnatoxin-G and pinnatoxin-G on Xenopus laevis oocytes having incorporated Torpedo muscle-type (α1)2βγδ nAChR into their plasma membranes. The peak amplitudes of the ACh-evoked current values (mean ± SEM; 5 oocytes per concentration) were normalized to control ACh-peak amplitudes recorded from the same oocyte to yield fractional response. Abbreviations: m/z: Mass-to-charge ratio; PnTX-A: Pinnatoxin-A; PnTX-G: Pinnatoxin-G; PnTX-G-palmitate: 28-O-palmitoyl pinnatoxin-G.