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Saxitoxin and neosaxitoxin as toxic principles of Alexandrium andersoni (Dinophyceae) from the Gulf of Naples, Italy
Toxicon 38 (2000) 1871±1877 www.elsevier.com/locate/toxicon
Short communication
Saxitoxin and neosaxitoxin as toxic principles of Alexandrium andersoni (Dinophyceae) from the Gulf of Naples, Italy Patrizia Ciminiello a, Ernesto Fattorusso a,*, Martino Forino a, Marina Montresor b a
Dipartimento di Chimica dell Sostanze Naturali, UniversitaÁ degli studi di Napoli ``Federico II'', via D. Montesano 49, 80131, Napoli, Italy b Stazione Zoologica ``A. Dohrn'', Villa Comunale, 80121 Napoli, Italy Received 19 November 1999; accepted 6 January 2000
Abstract A clonal culture of Alexandrium andersoni, obtained from germination of a resting cyst, collected from the Gulf of Naples, was found positive for PSP toxicity by mouse bioassay. The toxicity pro®le of this dino¯agellate consists mainly of toxins belonging to the saxitoxin class, in particular of Saxitoxin (STX) and Neosaxitoxin (NEO), as determined by a wide MS and 1H NMR analysis. This represents the ®rst report of the presence of A. andersoni in the Mediterranean Sea, as well as of its toxicity. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Alexandrium andersoni; PSP; Saxitoxin; Neosaxitoxin
1. Introduction Several species of the genus Alexandrium (Dinophyceae) produce potent toxins responsible for paralytic shell®sh poisoning (PSP), a neurological aection that has caused human illness for centuries and claimed hundreds of lives (Hallegrae, * Corresponding author Tel.: +39-81-7486-507/503; fax: +39-81-7486-552. E-mail address: [email protected] (E. Fattorusso). 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 0 9 9 - 4
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1993). Considerable progress has been achieved in the structural elucidation of PSP toxins and in their quantitation in a variety of producing and vectoral organisms. Since the structure of the parent compound saxitoxin (STX) was ®rst described, approximately two dozen naturally-occurring derivatives have been found among various organisms (Oshima, 1995). Toxigenic species typically produce more than a single toxin derivative, but no individual strain is known to synthesise the entire suite of compounds, and it has long been recognised that toxicity is variable within a single species (Alam et al., 1979; Shimizu, 1979; Hall, 1982; Maranda et al., 1985; Cembella et al., 1987). Single isolates can vary dramatically in toxin content under dierent growth conditions. The essential stability of the toxin pro®le and the dierences in toxin composition maintained among toxigenic isolates for all the PSP toxin-producing species argues forcefully for the dominance of genetic factors in determining and regulating toxin biosynthesis. Up to now, only two potentially toxic species of the genus Alexandrium have been reported for the Mediterranean Sea, A. minutum and A. tamarense. These species have been recorded at several locations spanning from the Spanish coasts, to the Adriatic Sea, till the eastern Mediterranean basin (Honsell et al., 1995; Labib and Halim, 1995; Sorokin et al., 1996; Garces et al., 1998; Forteza et al., 1998; Delgado et al., 1999). However, the toxigenic potential of these species in most of the geographic areas is still unknown, since toxicological analyses have been performed only in some cases. During an investigation on dino¯agellate cyst production in the Gulf of Naples (Tyrrhenian Sea, Mediterranean Sea), spherical, smooth-walled cysts, which germinated into Alexandrium andersoni Balech [Balech, 1990 ]239], were recorded in sediment traps during the summer months (Montresor et al., 1998). A. andersoni has been described from the eastern US coast (Balech, 1995) and, to our knowledge, the ®nding of this species in the Mediterranean Sea represents the ®rst report outside its type locality. This species was reported as non-toxic (Anderson et al., 1990; Balech, 1995); however, our preliminary investigation carried out on culture material from the Gulf of Naples has resulted positive for PSP using mouse bioassay. This positive response prompted the present investigation into the nature of the toxins produced by this species. 2. Material and methods 2.1. Cultures Alexandrium andersoni (culture SZN 12) was grown in 10 l polycarbonate bottles ®lled with 5 l K culture medium without silicates (Keller et al., 1987), at a temperature of about 208C and a photon ¯ux density of 100 mmol photons mÿ2 sÿ1. Late exponentially growing cultures were concentrated with a tangential ¯ow ®ltration system, and than centrifuged to obtain a pellet which was stored at ÿ208C.
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2.2. Mouse assay Assessment of toxicity of samples was undertaken by the ocial mouse bioassay method (Gazzetta Uciale, 1990). This method involves intraperitoneal injection of mice with an acidi®ed extract of toxins and the determination of death time, which is then converted into PSP concentration. The toxicity is expressed in terms of mouse units (MU), where 1 MU is de®ned as the amount of toxin required to kill a male ddY mouse of 20 g body weight in 15 h. According to this de®nition 1 MU corresponds to 0.2 mg of STX. 2.3. Toxins extraction and puri®cation Toxins extraction has been performed by suspending cultures (40 ml with about 2108 cells) in an equal volume of acetic acid 0.5 N and by sonifying the cell suspension for 1 h at a setting of 6 AÊ, while the sample was in an ice-water bath. The obtained solution has been centrifuged (3000 g/min, 30 min, 258C) and the supernatant aspirated. The presence of toxins in the supernatant has been con®rmed by the ocial assay. The total toxins amount was approximately 1100 MU. After evaporating the solvent, toxic material has been dissolved in H2O and passed through a Toyopearl HW-40 SF column using water as eluent. Toxins emerged from the Toyopearl column between 40 and 70 ml of water. Toxic fraction was loaded on a Polysep GFC-P2000 column eluted with water; and ®nally a good separation was achieved on a ODS column with TFA (15 mM)/ CH3CN 1:1 as eluent, which allowed the isolation of STX (570 MU) and NeoSTX (320 MU) in pure forms. Spectral data of these toxins were identical to those of authentic samples. 2.4. Instruments NMR spectra were measured on a Bruker AMX-500 spectrometer in D2O as solvent (CHCl3 at d 7.67 was used as an internal standard). FAB-MS were obtained at 70 eV on a Kratos MS50 mass spectrometer. Medium-pressure liquid chromatography (MPLC) was performed on a BuÈchi 861 apparatus, while HPLC on a Varian 2510 apparatus, equipped with Waters 490 MS UV. 2.5. Detection of toxins During the chromatographic puri®cation, elution of the toxins has been monitored with a UV spectromonitor, by thin-layer chromatography (TLC) and by mouse assay. The wavelength of the UV spectromonitor was set at 208 nm. TLC was carried out on silica gel 60 plates (Merck, precoated) using pyridine± EtOAc±water±AcOH (75:25:15:30) as eluent. Toxin spots were detected under long wave UV after spraying 1% H2O2 and heating at 1308C for 10 min. Mouse
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bioassay was performed following the ocial method for the determination of paralytic shell®sh poisoning (Gazzetta Uciale, 1990). 3. Results and discussion 3.1. Taxonomy Vegetative cells of Alexandrium andersoni are round to slightly oval in shape (average length=20.9 mm, st. dev. 2.0; average width=19.7, st. dev. 2.1 mm; n = 60). Cells have a light-brown pigmentation, chloroplasts are radially arranged in the cytoplasm, and a large horse-shoe shaped nucleus is located in the equatorial part of the cell. The cell surface is smooth; plate 1 ' is connected with the apical pore plate and has a small ventral pore along the suture with plate 4'. This species is very close to A. minutum, from which it can be distinguished mainly by the shape of plates 60 (6th precingular plate) and S.a. (Sulcal anterior plate). In A. andersoni, plate 60 is much narrower in its posterior part, due to a remarkably short cingular border, and it is characterised by a marked obliquity of the posterior left margin, which corresponds to the inclined right margin of plate S.a.. The middle part of plate 60 is thus notably wider than its posterior one. The sulcal anterior plate of A. andersoni diers from that of A. minutum for its trapezoidal shape due to its markedly oblique right margin. 3.2. Toxins analysis The cultured cells were extracted with mild acetic acid and tested for PSP analysis using mouse bioassay. The sample produced visual symptoms in the mice resembling the eect of paralytic shell®sh poisoning (PSP) produced in some species by the dino¯agellate Alexandrium, such as convulsion and jumping before death. Death occurred within a few minutes after injection. The extract was passed through several chromatographic columns, thus allowing the separation of the toxic material into two individual components, STX and NEO (Fig. 1). The amounts of toxins thus puri®ed were approximately 570 and 320 MU, respectively. Their identi®cation has been carried out by a wide NMR and mass-spectrometry analysis. The fast atom bombardment mass spectra (FAB-MS) of the puri®ed toxins were measured in glycerol as a matrix through a magnetic scan from 25 to 500 mass units. STX sample exhibited an ion peak at m/z = 300 and 282 by positive ion MS. The ion peak at 300 corresponds with the pseudomolecular ion (M+H)+ of authentic STX; while the fragment ion at 282 can be attributed to (M+HÿH2O)+. The fraction containing NEO showed an ion peak at m/z = 316, which corresponds with the pseudomolecular ion (M+H)+ of an authentic sample of the same toxin. 1 H NMR data were resolute in the identi®cation of the two PSP toxins. Proton
P. Ciminiello et al. / Toxicon 38 (2000) 1871±1877
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Fig. 1. Structures of paralytic shell®sh toxins.
chemical shifts of the two toxins were perfectly overlapping with those of authentic samples and with those reported in literature, thus allowing their unambiguous identi®cation. The 1H NMR values of the two toxins are reported in Table 1. The mouse bioassay performed on all the fractions emerging from the Table 1 1 H NMR chemical shifts of STX and NEO in D2O STX
NEO
C-H No.
d
J
d
J
5 6 10
4.75 3.86 3.56 3.83 Exchanged 4.03 4.29
s 9.4; 5.0 10.0 10.0; 7.9
4.83 4.10 3.59 3.80 Exchanged 4.42 4.23
s 6.2; 5.9 m 9.9; 7.0
11 13 a 13 b
11.5; 5.0 11.5; 9.4
11.7; 5.9 11.7; 6.2
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chromatographic columns evidenced the presence of another toxic fraction. However, the limited amount of the toxins in the sample enabled us to establish if they were known or novel PSP toxins. Our studies, however, have clearly established the toxicity of Alexandrium andersoni from the Gulf of Naples. Even if the analysis of the PSP content of this dino¯agellate is not complete yet, a striking dierence, however, already emerges from this analysis, as the Gulf of Naples do not resemble any other isolate of the related A. minutum, characterised to date (Oshima et al., 1989; Sournia et al., 1991; Hallegrae et al., 1991; Ledoux et al., 1993; Franco et al., 1994). Strains from Taiwan, Australia, Portugal, and Spain all contain mostly GTX 1,4 toxins. An isolate from Morlay Bay, France, contained only GTX 2,3 and C 1,2. The most similar pro®les are those of several A. minutum from Marlborough Sounds and Tasman Bay, as well as from the Bay of Plenty, New Zealand, which did contain some NEO, STX, and GTX 1-4, even if the toxin content is dierent one to each other (MacKenzie and Berkett, 1997). These results are not surprising, as toxin composition is known to vary widely among dierent Alexandrium isolates, even if they conform to the same morphotype (Oshima et al., 1982; Cembella et al., 1987; Noguchi et al., 1990; Anderson et al., 1994). Now that the potential for PSP toxicity has been veri®ed, a more vigilant monitoring is required to protect both the health of the public and the shell®sh industry. More work is also needed to identify other saxitoxinproducing strains or organisms in the region. Acknowledgements This work is a result of research supported by MURST PRIN ``Chimica dei Composti Organici di Interesse Biologico'', Rome, Italy. NMR and FABMS spectra were performed at ``Centro di Ricerca Interdipartimentale di Analisi strumentale'', UniversitaÁ degli studi di Napoli ``Federico II''. The assistance of the sta is gratefully appreciated. References Alam, M.I., Hsu, C.P., Shimizu, Y., 1979. Comparison of toxins in three isolates of Gonyaulax lamarensis (Dinophyceae). J. Phycol 15 (1), 106±110. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990. Dynamics and physiology of saxitioxin production by the dino¯agellate Alexandrium spp. Marine Biology 104, 511±524. Anderson, D.M., Kulis, D.M., Doucette, G.J., Gallagher, J.C., Balech, E., 1994. Biogeography of toxic dino¯agellates in the genus Alexandrium from the northeastern United States and Canada. Mar. Biol. 120, 467±478. Balech, E., 1995. The Genus Alexandrium Halim (Dino¯agellata). Sherkin Island Marine Station, Sherkin Island, Co. Cork, Ireland, p. 151. Cembella, A.D., Sullivan, J.J., Boyer, G.L., Taylor, F.J.R., Anderson, R.J., 1987. Variation in paralytic shell®sh toxin composition within the Protogonyaulax tamarensis/catenella species complex: red-tide dino¯agellates. Biochem. Systematics Ecol 15, 171±186. Delgado, M., Estrada, M., Camp, J., Fernandez, J.V., Santmarti, M., Lleti, M., 1999. Development of
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a toxic Alexandrium minutum Halim (Dinophyceae) bloom in the harbour of Sant Carles de la Rapita (Ebro Delta, northwestern Mediterranean). Scientia Marina 54, 1±7. Forteza, V., Quetglas, G., Delgado, M., Reyero, M.I., Fraga, S., Franco, J.M., Cacho, E. 1998. Toxic Alexandrium minutum bloom in Palma de Mallorca harbour (Balearic Islands, western Mediterranean). In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernamental Oceanographic Commission of UNESCO, pp. 58±59. Franco, J.R., Fernandez, P., Reguera, B., 1994. Toxin pro®les of natural population and cultures of Alexandrium minutum Halim from Galician (Spain) coastal waters. J. appl. Phycol 6, 272±279. Garces, E., Delgado, M., Vila, M., Camp, J. 1998. Alexandrium minutum bloom: in situ growth or accumulation? In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, pp. 167±170. Hall, S., 1982. Toxins and toxicity of Protogonyaulax from the northeast Paci®c. Ph.D. thesis. University of Alaska. Hallegrae, G.N., Bolch, C.J., Blackburn, S.I., Oshima, Y., 1991. Species of the toxigenic dino¯agellates genus Alexandrium in southeastern Australian waters. Bot. Mar. 34, 575±587. Hallegrae, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79±99. Honsell, G., Nichetto, P., Sidari, L., Tubaro, A., 1995. Toxic dino¯agellates in the Mediterranean Sea. Giornale Botanico Italiano 129, 390±403. Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R.L., 1987. Media for the culture of oceanic ultraphytoplankton. Journal of Phycology 23, 633±638. Labib, W., Halim, Y., 1995. Diel vertical migration and toxicity of Alexandrium minutum Halim red tide, in Alexandria, Egypt. Marine Life 5, 11±17. Ledoux, M., Bardouil, M., Fremy, J.M., Lassus, P., Murail, I., Bohec, M., 1993. Use of HPLC for toxin analysis of shell®sh contaminated by an Alexandrium minutum strain. Toxic Phytoplankton Blooms in the Sea, pp. 413±418. MacKenzie, L., Berkett, N., 1997. Cell morphology and PSP-toxin pro®les of Alexandrium minutum in the Marlborough Sounds, New Zealand. New Zealand Journal of Marine and Freshwater Research 31, 401±409. Maranda, L., Anderson, D.M., Shimizu, Y., 1985. Comparison of toxicity between population of Gonyaulax tamarensis of eastern North American waters. Estuar. estl. Shellf. Sci. 21, 401±410. Montresor, M., Zingone, A., Sarno, D., 1998. Dino¯agellate cyst production at a coastal Mediterranean site. Journal of Plankton Research 20, 2291±2312. Noguchi, T., Asakawa, M., Arakawa, O., Fukuyo, Y., Nishio, S., Tanno, K., Hashimoto, K., 1990. First occurrence of Alexandrium in Funka Bay, Hokkaido, along with its unique toxin composition. Toxic Marine Phytoplankton, pp. 493±498. Oshima, Y., Hayaskawa, T., Hashimoto, M., Kotaki, Y., Yasumoto, T., 1982. Classi®cation of Protogonyaulax tamarensis from northern Japan into three strains by toxin composition. Bull. Japan Soc. Sci. Fish 48, 851±854. Oshima, Y., Hirota, M., Yasumoto, T., Hallagrae, G., Blackburn, S., Steens, D., 1989. Production of paralytic shell®sh toxins by the dino¯agellate Alexandrium minutum Halim from Australia. Nippon Suisan Gakkaishi 55, 925. Oshima, Y. 1995. Post-column derivatization HPLC methods for paralytic shell®sh poisoning. In: Hallegrae, G.M., Anderson, D.M., Cembella, A.D., Enevoldsen, H.O. (Eds.), Manual of Harmful Marine Microalgae. Intergovernmental Oceanographic Commission of UNESCO, pp. 81±94. Shimizu, Y. 1979. Developments in the study of paralytic shell®sh toxins. In: Taylor, D.L., Seager, H.H. (Eds.), toxic dino¯agellate blooms. Elsevier, New York, pp. 321±326. Sorokin, Y.I., Sorokin, P.Y., Ravagnan, G., 1996. On an extremely dense bloom of the dino¯agellate Alexandrium tamarense in lagoons of the Po river delta: impact on the environment. J. Sea Res. 35, 251±255. Sournia, A., Belin, C., Berland, B., Erard-le Denn, E., Gentien, P., Grzebyk, D., Marcaillou-la Baut, C., Lassus, P., Partensky F., 1991. Alexandrium minutum (Dinophycees). In: Le Phytoplancton Nuisible des Cotes de France, de la Biologie a la Prevention, pp. 83±90. Centre National de La Recherche Scienti®que, Service de la Documentation et des Publications, IFREMERS.
Short communication
Saxitoxin and neosaxitoxin as toxic principles of Alexandrium andersoni (Dinophyceae) from the Gulf of Naples, Italy Patrizia Ciminiello a, Ernesto Fattorusso a,*, Martino Forino a, Marina Montresor b a
Dipartimento di Chimica dell Sostanze Naturali, UniversitaÁ degli studi di Napoli ``Federico II'', via D. Montesano 49, 80131, Napoli, Italy b Stazione Zoologica ``A. Dohrn'', Villa Comunale, 80121 Napoli, Italy Received 19 November 1999; accepted 6 January 2000
Abstract A clonal culture of Alexandrium andersoni, obtained from germination of a resting cyst, collected from the Gulf of Naples, was found positive for PSP toxicity by mouse bioassay. The toxicity pro®le of this dino¯agellate consists mainly of toxins belonging to the saxitoxin class, in particular of Saxitoxin (STX) and Neosaxitoxin (NEO), as determined by a wide MS and 1H NMR analysis. This represents the ®rst report of the presence of A. andersoni in the Mediterranean Sea, as well as of its toxicity. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Alexandrium andersoni; PSP; Saxitoxin; Neosaxitoxin
1. Introduction Several species of the genus Alexandrium (Dinophyceae) produce potent toxins responsible for paralytic shell®sh poisoning (PSP), a neurological aection that has caused human illness for centuries and claimed hundreds of lives (Hallegrae, * Corresponding author Tel.: +39-81-7486-507/503; fax: +39-81-7486-552. E-mail address: [email protected] (E. Fattorusso). 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 0 9 9 - 4
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1993). Considerable progress has been achieved in the structural elucidation of PSP toxins and in their quantitation in a variety of producing and vectoral organisms. Since the structure of the parent compound saxitoxin (STX) was ®rst described, approximately two dozen naturally-occurring derivatives have been found among various organisms (Oshima, 1995). Toxigenic species typically produce more than a single toxin derivative, but no individual strain is known to synthesise the entire suite of compounds, and it has long been recognised that toxicity is variable within a single species (Alam et al., 1979; Shimizu, 1979; Hall, 1982; Maranda et al., 1985; Cembella et al., 1987). Single isolates can vary dramatically in toxin content under dierent growth conditions. The essential stability of the toxin pro®le and the dierences in toxin composition maintained among toxigenic isolates for all the PSP toxin-producing species argues forcefully for the dominance of genetic factors in determining and regulating toxin biosynthesis. Up to now, only two potentially toxic species of the genus Alexandrium have been reported for the Mediterranean Sea, A. minutum and A. tamarense. These species have been recorded at several locations spanning from the Spanish coasts, to the Adriatic Sea, till the eastern Mediterranean basin (Honsell et al., 1995; Labib and Halim, 1995; Sorokin et al., 1996; Garces et al., 1998; Forteza et al., 1998; Delgado et al., 1999). However, the toxigenic potential of these species in most of the geographic areas is still unknown, since toxicological analyses have been performed only in some cases. During an investigation on dino¯agellate cyst production in the Gulf of Naples (Tyrrhenian Sea, Mediterranean Sea), spherical, smooth-walled cysts, which germinated into Alexandrium andersoni Balech [Balech, 1990 ]239], were recorded in sediment traps during the summer months (Montresor et al., 1998). A. andersoni has been described from the eastern US coast (Balech, 1995) and, to our knowledge, the ®nding of this species in the Mediterranean Sea represents the ®rst report outside its type locality. This species was reported as non-toxic (Anderson et al., 1990; Balech, 1995); however, our preliminary investigation carried out on culture material from the Gulf of Naples has resulted positive for PSP using mouse bioassay. This positive response prompted the present investigation into the nature of the toxins produced by this species. 2. Material and methods 2.1. Cultures Alexandrium andersoni (culture SZN 12) was grown in 10 l polycarbonate bottles ®lled with 5 l K culture medium without silicates (Keller et al., 1987), at a temperature of about 208C and a photon ¯ux density of 100 mmol photons mÿ2 sÿ1. Late exponentially growing cultures were concentrated with a tangential ¯ow ®ltration system, and than centrifuged to obtain a pellet which was stored at ÿ208C.
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2.2. Mouse assay Assessment of toxicity of samples was undertaken by the ocial mouse bioassay method (Gazzetta Uciale, 1990). This method involves intraperitoneal injection of mice with an acidi®ed extract of toxins and the determination of death time, which is then converted into PSP concentration. The toxicity is expressed in terms of mouse units (MU), where 1 MU is de®ned as the amount of toxin required to kill a male ddY mouse of 20 g body weight in 15 h. According to this de®nition 1 MU corresponds to 0.2 mg of STX. 2.3. Toxins extraction and puri®cation Toxins extraction has been performed by suspending cultures (40 ml with about 2108 cells) in an equal volume of acetic acid 0.5 N and by sonifying the cell suspension for 1 h at a setting of 6 AÊ, while the sample was in an ice-water bath. The obtained solution has been centrifuged (3000 g/min, 30 min, 258C) and the supernatant aspirated. The presence of toxins in the supernatant has been con®rmed by the ocial assay. The total toxins amount was approximately 1100 MU. After evaporating the solvent, toxic material has been dissolved in H2O and passed through a Toyopearl HW-40 SF column using water as eluent. Toxins emerged from the Toyopearl column between 40 and 70 ml of water. Toxic fraction was loaded on a Polysep GFC-P2000 column eluted with water; and ®nally a good separation was achieved on a ODS column with TFA (15 mM)/ CH3CN 1:1 as eluent, which allowed the isolation of STX (570 MU) and NeoSTX (320 MU) in pure forms. Spectral data of these toxins were identical to those of authentic samples. 2.4. Instruments NMR spectra were measured on a Bruker AMX-500 spectrometer in D2O as solvent (CHCl3 at d 7.67 was used as an internal standard). FAB-MS were obtained at 70 eV on a Kratos MS50 mass spectrometer. Medium-pressure liquid chromatography (MPLC) was performed on a BuÈchi 861 apparatus, while HPLC on a Varian 2510 apparatus, equipped with Waters 490 MS UV. 2.5. Detection of toxins During the chromatographic puri®cation, elution of the toxins has been monitored with a UV spectromonitor, by thin-layer chromatography (TLC) and by mouse assay. The wavelength of the UV spectromonitor was set at 208 nm. TLC was carried out on silica gel 60 plates (Merck, precoated) using pyridine± EtOAc±water±AcOH (75:25:15:30) as eluent. Toxin spots were detected under long wave UV after spraying 1% H2O2 and heating at 1308C for 10 min. Mouse
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bioassay was performed following the ocial method for the determination of paralytic shell®sh poisoning (Gazzetta Uciale, 1990). 3. Results and discussion 3.1. Taxonomy Vegetative cells of Alexandrium andersoni are round to slightly oval in shape (average length=20.9 mm, st. dev. 2.0; average width=19.7, st. dev. 2.1 mm; n = 60). Cells have a light-brown pigmentation, chloroplasts are radially arranged in the cytoplasm, and a large horse-shoe shaped nucleus is located in the equatorial part of the cell. The cell surface is smooth; plate 1 ' is connected with the apical pore plate and has a small ventral pore along the suture with plate 4'. This species is very close to A. minutum, from which it can be distinguished mainly by the shape of plates 60 (6th precingular plate) and S.a. (Sulcal anterior plate). In A. andersoni, plate 60 is much narrower in its posterior part, due to a remarkably short cingular border, and it is characterised by a marked obliquity of the posterior left margin, which corresponds to the inclined right margin of plate S.a.. The middle part of plate 60 is thus notably wider than its posterior one. The sulcal anterior plate of A. andersoni diers from that of A. minutum for its trapezoidal shape due to its markedly oblique right margin. 3.2. Toxins analysis The cultured cells were extracted with mild acetic acid and tested for PSP analysis using mouse bioassay. The sample produced visual symptoms in the mice resembling the eect of paralytic shell®sh poisoning (PSP) produced in some species by the dino¯agellate Alexandrium, such as convulsion and jumping before death. Death occurred within a few minutes after injection. The extract was passed through several chromatographic columns, thus allowing the separation of the toxic material into two individual components, STX and NEO (Fig. 1). The amounts of toxins thus puri®ed were approximately 570 and 320 MU, respectively. Their identi®cation has been carried out by a wide NMR and mass-spectrometry analysis. The fast atom bombardment mass spectra (FAB-MS) of the puri®ed toxins were measured in glycerol as a matrix through a magnetic scan from 25 to 500 mass units. STX sample exhibited an ion peak at m/z = 300 and 282 by positive ion MS. The ion peak at 300 corresponds with the pseudomolecular ion (M+H)+ of authentic STX; while the fragment ion at 282 can be attributed to (M+HÿH2O)+. The fraction containing NEO showed an ion peak at m/z = 316, which corresponds with the pseudomolecular ion (M+H)+ of an authentic sample of the same toxin. 1 H NMR data were resolute in the identi®cation of the two PSP toxins. Proton
P. Ciminiello et al. / Toxicon 38 (2000) 1871±1877
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Fig. 1. Structures of paralytic shell®sh toxins.
chemical shifts of the two toxins were perfectly overlapping with those of authentic samples and with those reported in literature, thus allowing their unambiguous identi®cation. The 1H NMR values of the two toxins are reported in Table 1. The mouse bioassay performed on all the fractions emerging from the Table 1 1 H NMR chemical shifts of STX and NEO in D2O STX
NEO
C-H No.
d
J
d
J
5 6 10
4.75 3.86 3.56 3.83 Exchanged 4.03 4.29
s 9.4; 5.0 10.0 10.0; 7.9
4.83 4.10 3.59 3.80 Exchanged 4.42 4.23
s 6.2; 5.9 m 9.9; 7.0
11 13 a 13 b
11.5; 5.0 11.5; 9.4
11.7; 5.9 11.7; 6.2
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chromatographic columns evidenced the presence of another toxic fraction. However, the limited amount of the toxins in the sample enabled us to establish if they were known or novel PSP toxins. Our studies, however, have clearly established the toxicity of Alexandrium andersoni from the Gulf of Naples. Even if the analysis of the PSP content of this dino¯agellate is not complete yet, a striking dierence, however, already emerges from this analysis, as the Gulf of Naples do not resemble any other isolate of the related A. minutum, characterised to date (Oshima et al., 1989; Sournia et al., 1991; Hallegrae et al., 1991; Ledoux et al., 1993; Franco et al., 1994). Strains from Taiwan, Australia, Portugal, and Spain all contain mostly GTX 1,4 toxins. An isolate from Morlay Bay, France, contained only GTX 2,3 and C 1,2. The most similar pro®les are those of several A. minutum from Marlborough Sounds and Tasman Bay, as well as from the Bay of Plenty, New Zealand, which did contain some NEO, STX, and GTX 1-4, even if the toxin content is dierent one to each other (MacKenzie and Berkett, 1997). These results are not surprising, as toxin composition is known to vary widely among dierent Alexandrium isolates, even if they conform to the same morphotype (Oshima et al., 1982; Cembella et al., 1987; Noguchi et al., 1990; Anderson et al., 1994). Now that the potential for PSP toxicity has been veri®ed, a more vigilant monitoring is required to protect both the health of the public and the shell®sh industry. More work is also needed to identify other saxitoxinproducing strains or organisms in the region. Acknowledgements This work is a result of research supported by MURST PRIN ``Chimica dei Composti Organici di Interesse Biologico'', Rome, Italy. NMR and FABMS spectra were performed at ``Centro di Ricerca Interdipartimentale di Analisi strumentale'', UniversitaÁ degli studi di Napoli ``Federico II''. The assistance of the sta is gratefully appreciated. References Alam, M.I., Hsu, C.P., Shimizu, Y., 1979. Comparison of toxins in three isolates of Gonyaulax lamarensis (Dinophyceae). J. Phycol 15 (1), 106±110. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990. Dynamics and physiology of saxitioxin production by the dino¯agellate Alexandrium spp. Marine Biology 104, 511±524. Anderson, D.M., Kulis, D.M., Doucette, G.J., Gallagher, J.C., Balech, E., 1994. Biogeography of toxic dino¯agellates in the genus Alexandrium from the northeastern United States and Canada. Mar. Biol. 120, 467±478. Balech, E., 1995. The Genus Alexandrium Halim (Dino¯agellata). Sherkin Island Marine Station, Sherkin Island, Co. Cork, Ireland, p. 151. Cembella, A.D., Sullivan, J.J., Boyer, G.L., Taylor, F.J.R., Anderson, R.J., 1987. Variation in paralytic shell®sh toxin composition within the Protogonyaulax tamarensis/catenella species complex: red-tide dino¯agellates. Biochem. Systematics Ecol 15, 171±186. Delgado, M., Estrada, M., Camp, J., Fernandez, J.V., Santmarti, M., Lleti, M., 1999. Development of
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