Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells Francisco Leira a,* , Ana G. Cabado a , Mercedes R. Vieytes b , Yolanda Roman c , Amparo Alfonso c , Luis M. Botana c , Takeshi Yasumoto d , Claudia Malaguti e , Gian P. Rossini e a ANFACO-CECOPESCA, Campus Universitario de Vigo, 36310 Vigo, Spain b Dpto. de Fisiologõ Âa Animal, Facultad de Veterinaria de Lugo, 27002 Lugo, Spain c Dpto. de Farmacologõ Âa, Facultad de Veterinaria de Lugo, 27002 Lugo, Spain d Japan Food Research Laboratories, Tama Laboratories, 6-11-10 Nagayama, Tama, Tokyo 206-0025, Japan e Dpto. di Scienze Biomediche, Universita  di Modena e Reggio Emilia, Via Campi 287, I-41100 Modena, Italy Received 22 January 2002; accepted 6 March 2002 Abstract Pectenotoxins are a group of marine toxins produced by dino¯agellates and formerly included within the group of diarrhetic shell®sh 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 ®rst isolated from toxic scallops. Results obtained demonstrate an speci®c 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 speci®c 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 in¯ux was ®rst activated, the toxin addition signi®cantly decreased the following calcium in¯ux. In these cells, pectenotoxin-6 only modi®es cAMP adenosine 3 0 ,5 0 -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. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Pectenotoxin-6; Cytotoxicity; F-actin; G-actin; Neuroblastoma; Apoptosis 1. Introduction Pectenotoxins PTXs) are a group of cyclic polyether macrolide compounds from marine origin ®rst isolated from toxic shell®sh by Yasumoto et al. [1]. PTXs have been classically included within DSP diarrhetic shell®sh poison) because of their phytoplanktonic origin and lipo- philic nature, thus, being co-extracted with diarrhogenic toxins okadaic acid, dinophysistoxins) from contaminated shell®sh. 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 shell®sh. PTX2 is suspected to be the precursor toxin of the whole PTXs through bio- transformation processes which take place in the digestive glands of bivalves. During last years, PTXs have been isolated from shell- ®sh of different culture areas in Europe, Asia and Oceania [4±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 mam- mals [7,8]. However, toxicological data are very scarce and further studies on the effects of PTXs are urgently required. Biochemical Pharmacology 63 2002) 1979±1988 0006-2952/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII:S0006-295202)00993-0 * Corresponding author. Tel.: 34-986469303; fax: 34-986469269. E-mail address: ¯eira@anfaco.es F. Leira). Abbreviations: DSP, diarrhetic shell®sh poison; PTXs, pectenotoxins; PTX6, pectenotoxin-6; PTX1, pectenotoxin-1; PTX2, pectenotoxin-2; EDTA, ethylenediaminetetracetic acid; SDS±PAGE, sodium dodecyl sulfate±polyacrilamide gel electrophoresis; PBS, phosphate buffered saline; cAMP, adenosine 3 0 ,5 0 -cyclic monophosphate.