Elsevier

Biochemical Pharmacology

Volume 63, Issue 11, 1 June 2002, Pages 1979-1988
Biochemical Pharmacology

Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells

https://doi.org/10.1016/S0006-2952(02)00993-0Get rights and content

Abstract

Pectenotoxins are a group of marine toxins produced by dinoflagellates and formerly included within the group of diarrhetic shellfish poison or toxins (DSP or DST) because of their physico-chemical properties. However, toxicological data on pectenotoxins are still very scarce and its mechanism of action is largely unknown, but toxicity in laboratory animals has been demonstrated by intraperitoneal injection. In this report, we present results of in vitro toxicological assessment of pectenotoxin-6, a derivative of the parental toxin pectenotoxin-2 first isolated from toxic scallops. Results obtained demonstrate an specific time- and dose-dependent depolymerization of F-actin in neuroblastoma cells exposed to pectenotoxin-6 (half-maximal effect about 700 nM at 24 hr). The change in the state of polymerization of actin was not accompanied by other major effects on specific signal transduction pathways or cell survival rate. Pectenotoxin-6 does not modify cytosolic calcium levels either in a calcium containing or calcium-free medium in human lymphocytes. Only when capacitative calcium influx was first activated, the toxin addition significantly decreased the following calcium influx. In these cells, pectenotoxin-6 only modifies cAMP (adenosine 3′,5′-cyclic monophosphate) levels in calcium-free conditions. In addition, no effect on cell attachment or apoptosis induction was observed at micromolar concentrations of pectenotoxin-6. Therefore, we conclude that cytoskeletal disruption is a key mechanism of PTX6-induced toxicity in eukaryotic cells.

Introduction

Pectenotoxins (PTXs) are a group of cyclic polyether macrolide compounds from marine origin first isolated from toxic shellfish by Yasumoto et al.[1]. PTXs have been classically included within DSP (diarrhetic shellfish poison) because of their phytoplanktonic origin and lipophilic nature, thus, being co-extracted with diarrhogenic toxins (okadaic acid, dinophysistoxins) from contaminated shellfish. Actually, eight different PTXs (PTX1 to 7 and PTX10) [2] and two new derivatives of PTX2 (PTX2 seco-acid and 7-epi-PTX2 seco-acid) [3] have been described and characterized mainly in shellfish. PTX2 is suspected to be the precursor toxin of the whole PTXs through biotransformation processes which take place in the digestive glands of bivalves.

During last years, PTXs have been isolated from shellfish of different culture areas in Europe, Asia and Oceania [4], [5], [6], thus, raising an increasing concern in Public Health authorities. In addition, a debate on the convenience of keeping PTXs within DSP has began since PTXs do not inhibit protein phosphatases nor induce diarrhea in mammals [7], [8]. However, toxicological data are very scarce and further studies on the effects of PTXs are urgently required. Most toxicological data available on PTXs (both in vivo and in vitro) have been obtained with PTX1, showing liver damage following intraperitoneal injection in mice [8] and morphological changes in freshly prepared hepatocytes [9], [10]. In addition, ld50 values in mice have been obtained for most PTXs, revealing the highest lethality for PTX2 [3], [11], which further supports the hypothesis of PTX2 as the parental compound of PTX group. Thus, successive oxidation of substituent in C18 in the digestive glands of bivalves would diminish the toxicity of PTXs. Additional data obtained with PTX1 showed no antibacterial or antifungal activity for this toxin [12], although its physico-chemical structure is similar to that of the polyether macrolide goniodomin A, a highly potent antifungal compound. PTX2 has been proven to induce lethality of brine shrimp (Artemia salina), as well as cytotoxic activity against several human cell lines, although significant differences were observed in the relative lc50 values obtained for each of them [13].

Apoptosis induction by PTXs has already been studied since apoptosis or programmed cell death is a common event in cells exposed to lipophilic phycotoxins [14], [15], [16], [17], [18], [19]. Primary cultures of rat and salmon hepatocytes exposed to PTX1 in the micromolar range showed rapid apoptotic changes [20], but no further studies concerning apoptotic activity of PTXs have been carried out in human cells. No additional data are available on acute and chronic effects of PTXs, and the mechanism of action of these toxins is currently unknown.

In this work, we present results obtained on in vitro toxicological evaluation of PTX6, an acidic PTX isolated from toxic scallops [11]. A wide array of morphological and biochemical indicators of toxicity have been assessed in different eukaryotic cells in order to obtain relevant information on the mechanism of action of PTX6. Key experiments have been confined to the most relevant cell lines due to the tiny amounts of pure toxin available. We demonstrate that PTX6 induces an specific time- and dose-dependent depolymerization of F-actin in neuroblastoma cells exposed to micromolar concentrations of PTX6. The change in the state of polymerization of actin was not accompanied by other major effects on specific signal transduction pathways or cell survival rate, thus, suggesting that cytoskeletal disruption is a key mechanism of PTX6-induced toxicity.

Section snippets

Materials

Peroxydase-linked anti-rabbit Ig antibodies, and the enhanced chemiluminescence (ECL) detection reagents were from Amersham Biosciences. Prestained molecular mass markers, and monoclonal anti-actin antibody from mouse were obtained from Sigma. The nitrocellulose membrane Protran B83 was obtained from Schleicher & Schuell. Thapsigargin, ionomycin and forskolin were from Alexis Corporation. FURA-2 AM, FlCRhR (recombinant fluorescein-and rhodamine-labeled protein kinase A) and Influx™ Pinocytic

Effect of PTX6 on F-actin cytoskeleton

Changes on F-actin have been evaluated since it is known that the actin cytoskeleton is targeted by several marine natural compounds including PTX2 [28]. As expected, neuroblastoma cells showed rapid morphological changes in response to PTX6. Results obtained by fluorimetric microplate assay (Fig. 1) demonstrated a time- and dose-dependent disruption of F-actin cytoskeleton in BE(2)-M17 cells exposed to PTX6 for 1–48 hr (half-maximal effect about 700 nM at 24 hr). Significant differences with

Discussion

During last years, PTXs have been reported in shellfish from different countries [4], [5], [6], thus, gaining an increasing interest for both shellfish producers and Public Health authorities. However, toxicological data on PTXs are still very scarce and its mechanism of action is currently unknown. In this report, we present the first data on cytotoxic activity of PTX6, a member of PTX group isolated from toxic scallops [11]. Results reported in this work provide initial evidence of the

Acknowledgements

This work was funded with grants REN2001-2959-C04-03/MAR and FEDER-CICYT-1FD97-0153 from Ministerio de Ciencia y Tecnologı́a (SPAIN), Xunta Galicia (PGIDT99INN26101 and PGIDT00MAR26101PR), and MCYT(DGI) BMC2000-0441. The authors thank technical assistance of CACTI from Universidad de Vigo (Spain) in the experiments with confocal microscope.

References (43)

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